Compositions and methods

ABSTRACT

Disclosed herein are therapeutic compositions containing non-pathogenic, germination-competent bacterial spores, for the prevention, control, and treatment of gastrointestinal diseases, disorders and conditions and for general nutritional health.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/884,655 filed Oct. 15, 2015, (allowed), which is a continuation ofU.S. application Ser. No. 14/313,828 filed Jun. 24, 2014, now U.S. Pat.No. 9,180,147, issued Nov. 10, 2015, which is a divisional of U.S.application Ser. No. 14/197,044, filed Mar. 4, 2014, now U.S. Pat. No.9,011,834, issued Apr. 21, 2015, which is a continuation ofInternational Application No. PCT/US2014/014745, filed Feb. 4, 2014,which claims priority to U.S. Provisional Application No. 61/760,584,filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,585,filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,574,filed Feb. 4, 2013, and U.S. Provisional Application No. 61/760,606,filed Feb. 4, 2013, and U.S. Provisional Application No. 61/926,918,filed Jan. 13, 2014. These applications are all incorporated byreference in their entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing with 2043 sequencessubmitted electronically as a text file named36011_US_Sequence_Listing.txt, created on Jan. 12, 2017, with a size of4,324,673 bytes. The sequence listing is incorporated by reference.

BACKGROUND

Mammals are colonized by microbes in the gastrointestinal (GI) tract, onthe skin, and in other epithelial and tissue niches such as the oralcavity, eye surface and vagina. The gastrointestinal tract harbors anabundant and diverse microbial community. It is a complex system,providing an environment or niche for a community of many differentspecies or organisms, including diverse strains of bacteria. Hundreds ofdifferent species may form a commensal community in the GI tract in ahealthy person, and this complement of organisms evolves from the timeof birth to ultimately form a functionally mature microbial populationby about 3 years of age. Interactions between microbial strains in thesepopulations and between microbes and the host, e.g. the host immunesystem, shape the community structure, with availability of andcompetition for resources affecting the distribution of microbes. Suchresources may be food, location and the availability of space to grow ora physical structure to which the microbe may attach. For example, hostdiet is involved in shaping the GI tract flora.

A healthy microbiota provides the host with multiple benefits, includingcolonization resistance to a broad spectrum of pathogens, essentialnutrient biosynthesis and absorption, and immune stimulation thatmaintains a healthy gut epithelium and an appropriately controlledsystemic immunity. In settings of ‘dysbiosis’ or disrupted symbiosis,microbiota functions can be lost or deranged, resulting in increasedsusceptibility to pathogens, altered metabolic profiles, or induction ofproinflammatory signals that can result in local or systemicinflammation or autoimmunity. Thus, the intestinal microbiota plays asignificant role in the pathogenesis of many diseases and disorders,including a variety of pathogenic infections of the gut. For instance,subjects become more susceptible to pathogenic infections when thenormal intestinal microbiota has been disturbed due to use ofbroad-spectrum antibiotics. Many of these diseases and disorders arechronic conditions that significantly decrease a subject's quality oflife and can be ultimately fatal.

Manufacturers of probiotics have asserted that their preparations ofbacteria promote mammalian health by preserving the natural microflorain the GI tract and reinforcing the normal controls on aberrant immuneresponses. See, e.g., U.S. Pat. No. 8,034,601. Probiotics, however, havebeen limited to a very narrow group of genera and a correspondinglylimited number of species; as such, they do not adequately replace themissing natural microflora of the GI tract in many situations.

Thus practitioners have a need for a method of populating a subject'sgastrointestinal tract with a diverse and useful selection of microbiotain order to alter a dysbiosis.

Therefore, in response to the need for durable, efficient, and effectivecompositions and methods for treatment of GI diseases by way ofrestoring or enhancing microbiota functions, we address these and othershortcomings of the prior art by providing compositions and methods fortreating subjects.

SUMMARY OF THE INVENTION

Disclosed herein are therapeutic compositions containing non-pathogenic,germination-competent bacterial spores, for the prevention, control, andtreatment of gastrointestinal diseases, disorders and conditions and forgeneral nutritional health. These compositions are advantageous in beingsuitable for safe administration to humans and other mammalian subjectsand are efficacious in numerous gastrointestinal diseases, disorders andconditions and in general nutritional health.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic of 16S rRNA gene and denotes thecoordinates of hypervariable regions 1-9 (V1-V9). Coordinates of V1-V9are 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173,1243-1294, and 1435-1465 respectively, based on numbering using E. colisystem of nomenclature defined by Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene (16S rRNA) from Escherichia coli,PNAS 75(10):4801-4805 (1978). FIG. 1B highlights in bold the nucleotidesequences for each hypervariable region in the exemplary reference E.coli 16S sequence described by Brosius et al. FIG. 1B discloses SEQ IDNO: 2043.

FIG. 2 shows a photograph of a CsCl gradient demonstrating the sporeseparation from other residual habitat material.

FIG. 3 shows three phase contrast image demonstrating the progressiveenrichment of spores from a fecal suspension; ethanol treated, CsClpurified spore preparation; and an ethanol treated, CsCl purified,sucrose purified spore preparation.

FIG. 4 shows a set of survival curves demonstrating efficacy of thespore population in a mouse prophylaxis model of C. difficile.

FIG. 5 provides a set of survival curves demonstrating efficacy of thespore population in a hamster relapse prevention model of C. difficile.

FIG. 6 demonstrates the cell viability under a variety of ethanol andheat treatments for varying lengths of time.

FIG. 7 demonstrates cell survivability from four donor fecal samplesafter heat treatment at 60 C for 5 minutes.

FIG. 8 demonstrates that ethanol reduces both anaerobic and aerobicbacterial species by several orders of magnitude in seconds.

FIG. 9 demonstrates the spore concentration of fecal donations frommultiple donors over time.

FIG. 10 shows the strong correlation and linear correspondence betweenthe measurement of DPA concentration by a coupled fluorescence assay andthe viable spore colony forming units

FIG. 11 demonstrates the effect on various germination treatments on theability to cultivate vegetative bacteria from a spore population.

FIG. 12 demonstrates the increase in bacterial diversity from using agerminant treatment to grow vegetative bacteria from spore populations.

FIG. 13 demonstrates the role of heat activation at various temperatureson spores from three different donor fecal samples.

FIG. 14 demonstrates a lysozyme treatment with heat activation improvesgermination at most temperatures.

FIG. 15 demonstrates spore concentrations present in a fecal samplegrown on various medias.

FIG. 16 demonstrates similar spore production from incubating plates for2 and 7 days after a spore population was germinated on plates withvarious medias.

FIG. 17 demonstrates the protective efficacy of the spore population inmice challenged with C. difficile as measured by the change in weight ofmice over the course of the experiment. Each plot tracks the change inthe individual mouse's weight relative to day −1 over the course of theexperiment. The number of deaths over the course of the experiment isindicated at the top of the chart and demonstrated by a line terminationprior to day 6. The top panels (from left to right) are the vehiclecontrol arm, the fecal suspension arm, and the untreated naive controlarm, while the bottom panels are the ethanol treated, gradient purifiedspore preparation; the ethanol treated, gradient purified, “germinable”spore preparation, and ethanol treated, gradient purified,“sporulatable” preparation.

FIG. 18 demonstrates the microbial diversity measured in the ethanoltreated spore treatment sample and patient pre- and post-treatmentsamples. Total microbial diversity is defined using the Chao1Alpha-Diversity Index and is measured at the same genomic samplingdepths to confirm adequate sequence coverage to assay the microbiome inthe target samples. The patient pretreatment (purple) harbored amicrobiome that was significantly reduced in total diversity as comparedto the ethanol treated spore treatment (red) and patient post treatmentat days 5 (blue), 14 (orange), and 25 (green).

FIG. 19 demonstrates how the patient microbial ecology is shifted bytreatment with an ethanol treated spore treatment from a dysbiotic stateto a state of health. Principle coordinates analysis based on the totaldiversity and structure of the microbiome (Bray Curtis Beta Diversity)of the patient pre- and post-treatment delineates that the combinationof engraftment of the OTUs from the spore treatment and the augmentationof the patient microbial ecology leads to a microbial ecology that isdistinct from both the pretreatment microbiome and the ecology of theethanol treated spore treatment.

FIG. 20 demonstrates the augmentation of bacteroides species in patientstreated with the spore population. Comparing the number of Bacteroidescolonies from fecal suspensions pre-treatment and in week 4 posttreatment reveals an increase of 4 logs or greater. Colonies wereenumerated by serial dilution and plating on Bacteroides Bile Esculinagar which is highly selective for the B. fragilis group. Species weredetermined by 16S full-length sequence identification.

FIG. 21 demonstrates the increase in number of species engrafting andspecies augmenting in patient's microbiomes after treatment with anethanol-treated spore population. Relative abundance of species thatengrafted or augmented as described were determined based on the numberof 16S sequence reads. Each plot is from a different patient treatedwith the ethanol-treated spore population for recurrent C. difficile.

The figures depict various embodiments of the present invention forpurposes of illustration only. One skilled in the art will readilyrecognize from the following discussion that alternative embodiments ofthe structures and methods illustrated herein may be employed withoutdeparting from the principles of the invention described herein.

DESCRIPTION OF THE TABLES

Table 1.

List of Operational Taxonomic Units (OTU) with taxonomic assignmentsmade to Genus, Species, and Phylogenetic Clade. Clade membership ofbacterial OTUs is based on 16S sequence data. Clades are defined basedon the topology of a phylogenetic tree that is constructed fromfull-length 16S sequences using maximum likelihood methods familiar toindividuals with ordinary skill in the art of phylogenetics. Clades areconstructed to ensure that all OTUs in a given clade are: (i) within aspecified number of bootstrap supported nodes from one another, and (ii)within 5% genetic similarity. OTUs that are within the same clade can bedistinguished as genetically and phylogenetically distinct from OTUs ina different clade based on 16S-V4 sequence data, while OTUs fallingwithin the same clade are closely related. OTUs falling within the sameclade are evolutionarily closely related and may or may not bedistinguishable from one another using 16S-V4 sequence data. Members ofthe same clade, due to their evolutionary relatedness, play similarfunctional roles in a microbial ecology such as that found in the humangut. Compositions substituting one species with another from the sameclade are likely to have conserved ecological function and therefore areuseful in the present invention. All OTUs are denoted as to theirputative capacity to form spores and whether they are a Pathogen orPathobiont (see Definitions for description of “Pathobiont”). NIAIDPriority Pathogens are denoted as ‘Category-A’, ‘Category-B’, or‘Category-C’, and Opportunistic Pathogens are denoted as ‘OP’. OTUs thatare not pathogenic or for which their ability to exist as a pathogen isunknown are denoted as ‘N’. The ‘SEQ ID Number’ denotes the identifierof the OTU in the Sequence Listing File and ‘Public DB Accession’denotes the identifier of the OTU in a public sequence repository.

Table 2 contains bacterial OTUs identified from the 16s analysis of theethanol treated spore population before and after a CsCl gradientpurification.

Table 3 contains the mortality and weight change of mice treated with adonor fecal suspension and an ethanol and/or heat-treated sporepreparation at various dilutions,

Table 4 contains OTUs identified from spore forming species generated bypicking colonies from a spore preparation involving various heattreatments

Table 5 contains OTUs not identified in untreated fecal slurries, butidentified in ethanol treated or heat treated spore populations.

Table 6 contains OTUs identified from an ethanol treated sporepopulation isolated from a microbiome sample from donor A.

Table 7 contains OTUs identified from an ethanol treated sporepopulation isolated from a microbiome sample from donor B.

Table 8 contains OTUs identified from an ethanol treated sporepopulation isolated from a microbiome sample from donor C.

Table 9 contains OTUs identified from an ethanol treated sporepopulation isolated from a microbiome sample from donor D.

Table 10 contains OTUs identified from an ethanol treated sporepopulation isolated from a microbiome sample from donor E.

Table 11 contains OTUs identified from an ethanol treated sporepopulation isolated from a microbiome sample from donor F.

Table 12 contains OTUs identified from growing ethanol treated sporepopulations on various media types.

Table 13. Species identified as “germinable” and “sporulatable” bycolony picking approach

Table YYY. Species identified as “germinable” using 16S-V4 NGS approach.

Table ZZZ. Species identified as “sporulatable” using 16s-V4 NGSapproach.

Table AC shows spore content data from 3 different ethanol treated sporepreparations used to successfully treat 3 patients suffering fromrecurrent C. difficile infection.

Table AD. DPA doses in Table AC when normalized to 4×10⁵ SCFU per dose

Table GB. OTUs detected by a minimum of ten 16S-V4 sequence reads in atleast a one ethanol treated spore preparation (pan-microbiome). OTUsthat engraft in a treated patients and the percentage of patients inwhich they engraft are denoted, as are the clades, spore forming status,and Keystone OTU status. Starred OTUs occur in ≧80% of the ethanol prepsand engraft in 50% of the treated patients.

Table GC ranks the top 20 OTUs by CES with the further requirement thatan OTU must be shown to engraft to be a considered an element of a coreecology.

Table GD: Subsets of the Core Ecology tested in the C. difficile mousemodel

Table GE: Results of bacterial compositions tested in a C. difficilemouse model.

Table GF. OTUs and their clade assignments tested in ternarycombinations with results in the in vitro inhibition assay

Table ZA. Microbial compositions administered via oral gavage on Day −1

Table TAB. Population of OTUs on Days 2, 3 and 4 following dosing withMicrobial Compositions

Table TAC. Population of clades on Days 2, 3 and 4 following dosing withMicrobial Compositions

Table TAD. Mortality by experimental group in mice challenged with 104.5C. difficile spores on Day 0

DETAILED DESCRIPTION

Overview

Disclosed herein are therapeutic compositions containing non-pathogenic,germination-competent bacterial spores, for the prevention, control, andtreatment of gastrointestinal diseases, disorders and conditions and forgeneral nutritional health. These compositions are advantageous in beingsuitable for safe administration to humans and other mammalian subjectsand are efficacious in numerous gastrointestinal diseases, disorders andconditions and in general nutritional health. While spore-basedcompositions are known, these are generally prepared according tovarious techniques such as lyophilization or spray-drying of liquidbacterial cultures, resulting in poor efficacy, instability, substantialvariability and lack of adequate safety and efficacy.

It has now been found that populations of bacterial spores can beobtained from biological materials obtained from mammalian subjects,including humans. These populations are formulated into compositions asprovided herein, and administered to mammalian subjects using themethods as provided herein.

Definitions

“Microbiota” refers to the community of microorganisms that occur(sustainably or transiently) in and on an animal subject, typically amammal such as a human, including eukaryotes, archaea, bacteria, andviruses (including bacterial viruses i.e., phage).

“Microbiome” refers to the genetic content of the communities ofmicrobes that live in and on the human body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)), wherein “genetic content”includes genomic DNA, RNA such as ribosomal RNA, the epigenome,plasmids, and all other types of genetic information.

“Microbial Carriage” or simply “Carriage” refers to the population ofmicrobes inhabiting a niche within or on humans. Carriage is oftendefined in terms of relative abundance. For example, OTU1 comprises 60%of the total microbial carriage, meaning that OTU1 has a relativeabundance of 60% compared to the other OTUs in the sample from which themeasurement was made. Carriage is most often based on genomic sequencingdata where the relative abundance or carriage of a single OTU or groupof OTUs is defined by the number of sequencing reads that are assignedto that OTU/s relative to the total number of sequencing reads for thesample.

“Microbial Augmentation” or simply “augmentation” refers to theestablishment or significant increase of a population of microbes thatare (i) absent or undetectable (as determined by the use of standardgenomic and microbiological techniques) from the administeredtherapeutic microbial composition, (ii) absent, undetectable, or presentat low frequencies in the host niche (as example: gastrointestinaltract, skin, anterior-nares, or vagina) before the delivery of themicrobial composition, and (iii) are found after the administration ofthe microbial composition or significantly increase, for instance2-fold, 5-fold, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, or greaterthan 1×10⁸, in cases where they were present at low frequencies. Themicrobes that comprise an augmented ecology can be derived fromexogenous sources such as food and the environment, or grow out frommicro-niches within the host where they reside at low frequency.

The administration of the therapeutic microbial composition induces anenvironmental shift in the target niche that promotes favorableconditions for the growth of these commensal microbes. In the absence oftreatment with a therapeutic microbial composition, the host can beconstantly exposed to these microbes; however, sustained growth and thepositive health effects associated with the stable population ofincreased levels of the microbes comprising the augmented ecology arenot observed.

“Microbial Engraftment” or simply “engraftment” refers to theestablishment of OTUs comprising a therapeutic microbial composition ina target niche that are absent in the treated host prior to treatment.The microbes that comprise the engrafted ecology are found in thetherapeutic microbial composition and establish as constituents of thehost microbial ecology upon treatment. Engrafted OTUs can establish fora transient period of time, or demonstrate long-term stability in themicrobial ecology that populates the host post treatment with atherapeutic microbial composition. The engrafted ecology can induce anenvironmental shift in the target niche that promotes favorableconditions for the growth of commensal microbes capable of catalyzing ashift from a dysbiotic ecology to one representative of a health state.

“Ecological Niche” or simply “Niche” refers to the ecological space inwhich a an organism or group of organisms occupies. Niche describes howan organism or population or organisms responds to the distribution ofresources, physical parameters (e.g., host tissue space) and competitors(e.g., by growing when resources are abundant, and when predators,parasites and pathogens are scarce) and how it in turn alters those samefactors (e.g., limiting access to resources by other organisms, actingas a food source for predators and a consumer of prey).

“Dysbiosis” refers to a state of the microbiota of the gut or other bodyarea in a subject, including mucosal or skin surfaces in which thenormal diversity and/or function of the ecological network is disrupted.This unhealthy state can be due to a decrease in diversity, theovergrowth of one or more pathogens or pathobionts, symbiotic organismsable to cause disease only when certain genetic and/or environmentalconditions are present in a subject, or the shift to an ecologicalmicrobial network that no longer provides an essential function to thehost subject, and therefore no longer promotes health.

“Pathobionts” or “Opportunistic Pathogens” refers to symbiotic organismsable to cause disease only when certain genetic and/or environmentalconditions are present in a subject.

“Phylogenetic tree” refers to a graphical representation of theevolutionary relationships of one genetic sequence to another that isgenerated using a defined set of phylogenetic reconstruction algorithms(e.g. parsimony, maximum likelihood, or Bayesian). Nodes in the treerepresent distinct ancestral sequences and the confidence of any node isprovided by a bootstrap or Bayesian posterior probability, whichmeasures branch uncertainty.

“Operational taxonomic units,” “OTU” (or plural, “OTUs”) refer to aterminal leaf in a phylogenetic tree and is defined by a nucleic acidsequence, e.g., the entire genome, or a specific genetic sequence, andall sequences that share sequence identity to this nucleic acid sequenceat the level of species. In some embodiments the specific geneticsequence may be the 16S sequence or a portion of the 16S sequence. Inother embodiments, the entire genomes of two entities are sequenced andcompared. In another embodiment, select regions such as multilocussequence tags (MLST), specific genes, or sets of genes may begenetically compared. In 16S embodiments, OTUs that share ≧97% averagenucleotide identity across the entire 16S or some variable region of the16S are considered the same OTU (see e.g. Claesson M J, Wang Q,O'Sullivan O, Greene-Diniz R, Cole J R, Ros R P, and O'Toole P W. 2010.Comparison of two next-generation sequencing technologies for resolvinghighly complex microbiota composition using tandem variable 16S rRNAgene regions. Nucleic Acids Res 38: e200. Konstantinidis K T, Ramette A,and Tiedje J M. 2006. The bacterial species definition in the genomicera. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.). In embodimentsinvolving the complete genome, MLSTs, specific genes, or sets of genesOTUs that share ≧95% average nucleotide identity are considered the sameOTU (see e.g. Achtman M, and Wagner M. 2008. Microbial diversity and thegenetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440.Konstantinidis K T, Ramette A, and Tiedje J M. 2006. The bacterialspecies definition in the genomic era. Philos Trans R Soc Lond B BiolSci 361: 1929-1940.). OTUs are frequently defined by comparing sequencesbetween organisms. Generally, sequences with less than 95% sequenceidentity are not considered to form part of the same OTU. OTUs may alsobe characterized by any combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof. Such characterization employs, e.g., WGS data orawhole genome sequence.

“Residual habitat products” refers to material derived from the habitatfor microbiota within or on a human or animal. For example, microbiotalive in feces in the gastrointestinal tract, on the skin itself, insaliva, mucus of the respiratory tract, or secretions of thegenitourinary tract (i.e., biological matter associated with themicrobial community). Substantially free of residual habitat productsmeans that the bacterial composition no longer contains the biologicalmatter associated with the microbial environment on or in the human oranimal subject and is 100% free, 99% free, 98% free, 97% free, 96% free,or 95% free of any contaminating biological matter associated with themicrobial community. Residual habitat products can include abioticmaterials (including undigested food) or it can include unwantedmicroorganisms. Substantially free of residual habitat products may alsomean that the bacterial composition contains no detectable cells from ahuman or animal and that only microbial cells are detectable. In oneembodiment, substantially free of residual habitat products may alsomean that the bacterial composition contains no detectable viral(including bacterial viruses (i.e., phage)), fungal, mycoplasmalcontaminants. In another embodiment, it means that fewer than 1×10⁻²%,1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸ of the viable cellsin the bacterial composition are human or animal, as compared tomicrobial cells. There are multiple ways to accomplish this degree ofpurity, none of which are limiting. Thus, contamination may be reducedby isolating desired constituents through multiple steps of streaking tosingle colonies on solid media until replicate (such as, but not limitedto, two) streaks from serial single colonies have shown only a singlecolony morphology. Alternatively, reduction of contamination can beaccomplished by multiple rounds of serial dilutions to single desiredcells (e.g., a dilution of 10⁻⁸ or 10⁻⁹), such as through multiple10-fold serial dilutions. This can further be confirmed by showing thatmultiple isolated colonies have similar cell shapes and Gram stainingbehavior. Other methods for confirming adequate purity include geneticanalysis (e.g. PCR, DNA sequencing), serology and antigen analysis,enzymatic and metabolic analysis, and methods using instrumentation suchas flow cytometry with reagents that distinguish desired constituentsfrom contaminants.

“Clade” refers to the OTUs or members of a phylogenetic tree that aredownstream of a statistically valid node in a phylogenetic tree. Theclade comprises a set of terminal leaves in the phylogenetic tree thatis a distinct monophyletic evolutionary unit and that share some extentof sequence similarity.

In microbiology, “16S sequencing” or “16S-rRNA” or “16S” refers tosequence derived by characterizing the nucleotides that comprise the 16Sribosomal RNA gene(s). The bacterial 16S rDNA is approximately 1500nucleotides in length and is used in reconstructing the evolutionaryrelationships and sequence similarity of one bacterial isolate toanother using phylogenetic approaches. 16S sequences are used forphylogenetic reconstruction as they are in general highly conserved, butcontain specific hypervariable regions that harbor sufficient nucleotidediversity to differentiate genera and species of most bacteria.

The “V1-V9 regions” of the 16S rRNA refers to the first through ninthhypervariable regions of the 16S rRNA gene that are used for genetictyping of bacterial samples. These regions in bacteria are defined bynucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043,1117-1173, 1243-1294 and 1435-1465 respectively using numbering based onthe E. coli system of nomenclature. Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS75(10):4801-4805 (1978). In some embodiments, at least one of the V1,V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize anOTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU. A person of ordinary skill in the art canidentify the specific hypervariable regions of a candidate 16S rRNA bycomparing the candidate sequence in question to a reference sequence andidentifying the hypervariable regions based on similarity to thereference hypervariable regions, or alternatively, one can employ WholeGenome Shotgun (WGS) sequence characterization of microbes or amicrobial community.

The term “subject” refers to any animal subject including humans,laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows,sheep, goats, pigs, turkeys, and chickens), and household pets (e.g.,dogs, cats, and rodents). The subject may be suffering from a dysbiosis,including, but not limited to, an infection due to a gastrointestinalpathogen or may be at risk of developing or transmitting to others aninfection due to a gastrointestinal pathogen.

The term “phenotype” refers to a set of observable characteristics of anindividual entity. As example an individual subject may have a phenotypeof “health” or “disease”. Phenotypes describe the state of an entity andall entities within a phenotype share the same set of characteristicsthat describe the phenotype. The phenotype of an individual results inpart, or in whole, from the interaction of the entities genome and/ormicrobiome with the environment.

The term “Network Ecology” refers to a consortium of OTUs that co-occurin some number of subjects. As used herein, a “network” is definedmathematically by a graph delineating how specific nodes (i.e. OTUs) andedges (connections between specific OTUs) relate to one another todefine the structural ecology of a consortium of OTUs. Any given NetworkEcology will possess inherent phylogenetic diversity and functionalproperties. A Network Ecology can also be defined in terms of functionwhere for example the nodes would be comprised of elements such as, butnot limited to, enzymes, clusters of orthologous groups (COGS;http://www.ncbi.nlm.nih.gov/books/NBK21090/), or KEGG pathways(www.genome.jp/kegg/).

The terms “Network Class”, “Core Network” and “Core Network Ecology”refer to a group of network ecologies that in general arecomputationally determined to comprise ecologies with similarphylogenetic and/or functional characteristics. A Core Network thereforecontains important biological features, defined either phylogeneticallyor functionally, of a group (i.e., a cluster) of related networkecologies. One representation of a Core Network Ecology is a designedconsortium of microbes, typically non-pathogenic bacteria, thatrepresents core features of a set of phylogenetically or functionallyrelated network ecologies seen in many different subjects. In manyoccurrences, a Core Network, while designed as described herein, existsas a Network Ecology observed in one or more subjects. Core Networkecologies are useful for reversing or reducing a dysbiosis in subjectswhere the underlying, related Network Ecology has been disrupted.

The term “Keystone OTU” refers to one or more OTUs that are common tomany network ecologies and are members of networks ecologies that occurin many subjects (i.e. are pervasive) (FIG. 1). Due to the ubiquitousnature of Keystone OTUs, they are central to the function of networkecologies in healthy subjects and are often missing or at reduced levelsin subjects with disease. Keystone OTUs may exist in low, moderate, orhigh abundance in subjects.

The term “non-Keystone OTU” refers to an OTU that is observed in aNetwork Ecology and is not a keystone OTU.

The term “Phylogenetic Diversity” refers to the biodiversity present ina given Network Ecology or Core Network Ecology based on the OTUs thatcomprise the network. Phylogenetic diversity is a relative term, meaningthat a Network Ecology or Core Network that is comparatively morephylogenetically diverse than another network contains a greater numberof unique species, genera, and taxonomic families. Uniqueness of aspecies, genera, or taxonomic family is generally defined using aphylogenetic tree that represents the genetic diversity all species,genera, or taxonomic families relative to one another. In anotherembodiment phylogenetic diversity may be measured using the total branchlength or average branch length of a phylogenetic tree.

“Spore” or “endospore” refers to an entity, particularly a bacterialentity, which is in a dormant, non-vegetative and non-reproductivestage. Spores are generally resistant to environmental stress such asradiation, desiccation, enzymatic treatment, temperature variation,nutrient deprivation, and chemical disinfectants.

A “spore population” refers to a plurality of spores present in acomposition. Synonymous terms used herein include spore composition,spore preparation, ethanol treated spore fraction and spore ecology. Aspore population may be purified from a fecal donation, e.g. via ethanolor heat treatment, or a density gradient separation or any combinationof methods described herein to increase the purity, potency and/orconcentration of spores in a sample. Alternatively, a spore populationmay be derived through culture methods starting from isolated sporeformer species or spore former OTUs or from a mixture of such species,either in vegetative or spore form.

In one embodiment, the spore preparation comprises spore forming specieswherein residual non-spore forming species have been inactivated bychemical or physical treatments including ethanol, detergent, heat,sonication, and the like; or wherein the non-spore forming species havebeen removed from the spore preparation by various separations stepsincluding density gradients, centrifugation, filtration and/orchromatography; or wherein inactivation and separation methods arecombined to make the spore preparation. In yet another embodiment, thespore preparation comprises spore forming species that are enriched overviable non-spore formers or vegetative forms of spore formers. In thisembodiment, spores are enriched by 2-fold, 5-fold, 10-fold, 50-fold,100-fold, 1000-fold, 10,000-fold or greater than 10,000-fold compared toall vegetative forms of bacteria. In yet another embodiment, the sporesin the spore preparation undergo partial germination during processingand formulation such that the final composition comprises spores andvegetative bacteria derived from spore forming species.

A “germinant” is a material or composition or physical-chemical processcapable of inducing vegetative growth of a bacterium that is in adormant spore form, or group of bacteria in the spore form, eitherdirectly or indirectly in a host organism and/or in vitro.

A “sporulation induction agent” is a material or physical-chemicalprocess that is capable of inducing sporulation in a bacterium, eitherdirectly or indirectly, in a host organism and/or in vitro.

To “increase production of bacterial spores” includes an activity or asporulation induction agent. “Production” includes conversion ofvegetative bacterial cells into spores and augmentation of the rate ofsuch conversion, as well as decreasing the germination of bacteria inspore form, decreasing the rate of spore decay in vivo, or ex vivo, orto increasing the total output of spores (e.g. via an increase involumetric output of fecal material).

The “colonization” of a host organism includes the non-transitoryresidence of a bacterium or other microscopic organism. As used herein,“reducing colonization” of a host subject's gastrointestinal tract (orany other microbiotal niche) by a pathogenic bacterium includes areduction in the residence time of the pathogen in the gastrointestinaltract as well as a reduction in the number (or concentration) of thepathogen in the gastrointestinal tract or adhered to the luminal surfaceof the gastrointestinal tract. Measuring reductions of adherentpathogens may be demonstrated, e.g., by a biopsy sample, or reductionsmay be measured indirectly, e.g., by measuring the pathogenic burden inthe stool of a mammalian host.

A “combination” of two or more bacteria includes the physicalco-existence of the two bacteria, either in the same material or productor in physically connected products, as well as the temporalco-administration or co-localization of the two bacteria.

A “cytotoxic” activity or bacterium includes the ability to kill abacterial cell, such as a pathogenic bacterial cell. A “cytostatic”activity or bacterium includes the ability to inhibit, partially orfully, growth, metabolism, and/or proliferation of a bacterial cell,such as a pathogenic bacterial cell.

To be free of “non-comestible products” means that a bacterialcomposition or other material provided herein does not have asubstantial amount of a non-comestible product, e.g., a product ormaterial that is inedible, harmful or otherwise undesired in a productsuitable for administration, e.g., oral administration, to a humansubject. Non-comestible products are often found in preparations ofbacteria from the prior art.

As used herein the term “vitamin” is understood to include any ofvarious fat-soluble or water-soluble organic substances (non-limitingexamples include vitamin A, Vitamin B1 (thiamine), Vitamin B2(riboflavin), Vitamin B3 (niacin or niacinamide), Vitamin B5(pantothenic acid), Vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine,or pyridoxine hydrochloride), Vitamin B7 (biotin), Vitamin B9 (folicacid), and Vitamin B12 (various cobalamins; commonly cyanocobalamin invitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, K1 andK2 (i.e. MK-4, MK-7), folic acid and biotin) essential in minute amountsfor normal growth and activity of the body and obtained naturally fromplant and animal foods or synthetically made, pro-vitamins, derivatives,analogs.

As used herein, the term “minerals” is understood to include boron,calcium, chromium, copper, iodine, iron, magnesium, manganese,molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin,vanadium, zinc, or combinations thereof.

As used herein, the term “antioxidant” is understood to include any oneor more of various substances such as beta-carotene (a vitamin Aprecursor), vitamin C, vitamin E, and selenium) that inhibit oxidationor reactions promoted by Reactive Oxygen Species (“ROS”) and otherradical and non-radical species. Additionally, antioxidants aremolecules capable of slowing or preventing the oxidation of othermolecules. Non-limiting examples of antioxidants include astaxanthin,carotenoids, coenzyme Q10 (“CoQ10”), flavonoids, glutathione, Goji(wolfberry), hesperidin, lactowolfberry, lignan, lutein, lycopene,polyphenols, selenium, vitamin A, vitamin C, vitamin E, zeaxanthin, orcombinations thereof.

Compositions of the Invention

Disclosed herein are therapeutic compositions containing non-pathogenic,germination-competent bacterial spores, for the prevention, control, andtreatment of gastrointestinal diseases, disorders and conditions and forgeneral nutritional health. These compositions are advantageous in beingsuitable for safe administration to humans and other mammalian subjectsand are efficacious in numerous gastrointestinal diseases, disorders andconditions and in general nutritional health. While spore-basedcompositions are known, these are generally prepared according tovarious techniques such as lyophilization or spray-drying of liquidbacterial cultures, resulting in poor efficacy, instability, substantialvariability and lack of adequate safety and efficacy.

It has now been found that populations of bacterial spores can beobtained from biological materials obtained from mammalian subjects,including humans. These populations are formulated into compositions asprovided herein, and administered to mammalian subjects using themethods as provided herein.

Provided herein are therapeutic compositions containing a purifiedpopulation of bacterial spores. As used herein, the terms “purify”,“purified” and “purifying” refer to the state of a population (e.g., aplurality of known or unknown amount and/or concentration) of desiredbacterial spores, that have undergone one or more processes ofpurification, e.g., a selection or an enrichment of the desiredbacterial spore, or alternatively a removal or reduction of residualhabitat products as described herein. In some embodiments, a purifiedpopulation has no detectable undesired activity or, alternatively, thelevel or amount of the undesired activity is at or below an acceptablelevel or amount. In other embodiments, a purified population has anamount and/or concentration of desired bacterial spores at or above anacceptable amount and/or concentration. In other embodiments, the ratioof desired-to-undesired activity (e.g. spores compared to vegetativebacteria), has changed by 2-, 5-, 10-, 30-, 100-, 300-, 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, or greater than 1×10⁸. In other embodiments, thepurified population of bacterial spores is enriched as compared to thestarting material (e.g., a fecal material) from which the population isobtained. This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%,99.9999%, or greater than 99.999999% as compared to the startingmaterial.

In certain embodiments, the purified populations of bacterial sporeshave reduced or undetectable levels of one or more pathogenicactivities, such as toxicity, an ability to cause infection of themammalian recipient subject, an undesired immunomodulatory activity, anautoimmune response, a metabolic response, or an inflammatory responseor a neurological response. Such a reduction in a pathogenic activitymay be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% ascompared to the starting material. In other embodiments, the purifiedpopulations of bacterial spores have reduced sensory components ascompared to fecal material, such as reduced odor, taste, appearance, andumami.

Provided are purified populations of bacterial spores that aresubstantially free of residual habitat products. In certain embodiments,this means that the bacterial spore composition no longer contains asubstantial amount of the biological matter associated with themicrobial community while living on or in the human or animal subject,and the purified population of spores may be 100% free, 99% free, 98%free, 97% free, 96% free, or 95% free of any contamination of thebiological matter associated with the microbial community. Substantiallyfree of residual habitat products may also mean that the bacterial sporecomposition contains no detectable cells from a human or animal, andthat only microbial cells are detectable, in particular, only desiredmicrobial cells are detectable. In another embodiment, it means thatfewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸%of the cells in the bacterial composition are human or animal, ascompared to microbial cells. In another embodiment, the residual habitatproduct present in the purified population is reduced at least a certainlevel from the fecal material obtained from the mammalian donor subject,e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, orgreater than 99.9999%.

In one embodiment, substantially free of residual habitat products orsubstantially free of a detectable level of a pathogenic material meansthat the bacterial composition contains no detectable viral (includingbacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmalcontaminants, or a eukaryotic parasite such as a helminth.Alternatively, the purified spore populations are substantially free ofan acellular material, e.g., DNA, viral coat material, or non-viablebacterial material. Alternatively, the purified spore population mayprocessed by a method that kills, inactivates, or removes one or morespecific undesirable viruses, such as an enteric virus, includingnorovirus, poliovirus or hepatitis A virus.

As described herein, purified spore populations can be demonstrated bygenetic analysis (e.g., PCR, DNA sequencing), serology and antigenanalysis, microscopic analysis, microbial analysis including germinationand culturing, and methods using instrumentation such as flow cytometrywith reagents that distinguish desired bacterial spores fromnon-desired, contaminating materials.

Exemplary biological materials include fecal materials such as feces ormaterials isolated from the various segments of the small and largeintestines. Fecal materials are obtained from a mammalian donor subject,or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400,500, 750, 1000 or from greater than 1000 donors, where such materialsare then pooled prior to purification of the desired bacterial spores.In another embodiment, fecal materials can be obtained from a singledonor subject over multiple times and pooled from multiple samples e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 32, 35, 40, 45, 48, 50,100 samples from a single donor.

In alternative embodiments, the desired bacterial spores are purifiedfrom a single fecal material sample obtained from a single donor, andafter such purification are combined with purified spore populationsfrom other purifications, either from the same donor at a differenttime, or from one or more different donors, or both.

Mammalian donor subjects are generally of good health and havemicrobiota consistent with such good health. Often, the donor subjectshave not been administered antibiotic compounds within a certain periodprior to the collection of the fecal material. In certain embodiments,the donor subjects are not obese or overweight, and may have body massindex (BMI) scores of below 25, such as between 18.5 and 24.9. In otherembodiments, the donor subjects are not mentally ill or have no historyor familial history of mental illness, such as anxiety disorder,depression, bipolar disorder, autism spectrum disorders, schizophrenia,panic disorders, attention deficit (hyperactivity) disorders, eatingdisorders or mood disorders. In other embodiments, the donor subjects donot have irritable bowel disease (e.g., crohn's disease, ulcerativecolitis), irritable bowel syndrome, celiac disease, colorectal cancer ora family history of these diseases. In other embodiments, donors havebeen screened for blood borne pathogens and fecal transmissiblepathogens using standard techniques known to one in the art (e.g.nucleic acid testing, serological testing, antigen testing, culturingtechniques, enzymatic assays, assays of cell free fecal filtrateslooking for toxins on susceptible cell culture substrates).

In some embodiments, donors are also selected for the presence ofcertain genera and/or species that provide increased efficacy oftherapeutic compositions containing these genera or species. In otherembodiments, donors are preferred that produce relatively higherconcentrations of spores in fecal material than other donors. In furtherembodiments, donors are preferred that provide fecal material from whichspores having increased efficacy are purified; this increased efficacyis measured using in vitro or in animal studies as described below. Insome embodiments, the donor may be subjected to one or more pre-donationtreatments in order to reduce undesired material in the fecal material,and/or increase desired spore populations.

It is advantageous to screen the health of the donor subject prior toand optionally, one or more times after, the collection of the fecalmaterial. Such screening identifies donors carrying pathogenic materialssuch as viruses (HIV, hepatitis, polio) and pathogenic bacteria.Post-collection, donors are screened about one week, two weeks, threeweeks, one month, two months, three months, six months, one year or morethan one year, and the frequency of such screening may be daily, weekly,bi-weekly, monthly, bi-monthly, semi-yearly or yearly. Donors that arescreened and do not test positive, either before or after donation orboth, are considered “validated” donors.

Solvent Treatments.

To purify the bacterial spores, the fecal material is subjected to oneor more solvent treatments. A solvent treatment is a miscible solventtreatment (either partially miscible or fully miscible) or an immisciblesolvent treatment. Miscibility is the ability of two liquids to mix witheach to form a homogeneous solution. Water and ethanol, for example, arefully miscible such that a mixture containing water and ethanol in anyratio will show only one phase. Miscibility is provided as a wt/wt %, orweight of one solvent in 100 g of final solution. If two solvents arefully miscible in all proportions, their miscibility is 100%. Providedas fully miscible solutions with water are alcohols, e.g., methanol,ethanol, isopropanol, butanol, propanediol, butanediol, etc. Thealcohols can be provided already combined with water; e.g., a solutioncontaining 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 89%, 85%, 90%, 95% or greater than 95%. Other solvents are onlypartially miscible, meaning that only some portion will dissolve inwater. Diethyl ether, for example, is partially miscible with water. Upto 7 grams of diethyl ether will dissolve in 93 g of water to give a 7%(wt/wt %) solution. If more diethyl ether is added, a two-phase solutionwill result with a distinct diethyl ether layer above the water. Otherpartially miscible materials include ethers, propanoate, butanoate,chloroform, dimethoxyethane, or tetrahydrofuran. In contrast, an oilsuch as an alkane and water are immiscible and form two phases. Further,immiscible treatments are optionally combined with a detergent, eitheran ionic detergent or a non-ionic detergent. Exemplary detergentsinclude Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, or apolyol. The solvent treatment steps reduces the viability of non-sporeforming bacterial species by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90%, 95%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and it mayoptionally reduce the viability of contaminating protists, parasitesand/or viruses.

Chromatography Treatments.

To purify spore populations, the fecal materials are subjected to one ormore chromatographic treatments, either sequentially or in parallel. Ina chromatographic treatment, a solution containing the fecal material iscontacted with a solid medium containing a hydrophobic interactionchromatographic (HIC) medium or an affinity chromatographic medium. Inan alternative embodiment, a solid medium capable of absorbing aresidual habitat product present in the fecal material is contacted witha solid medium that adsorbs a residual habitat product. In certainembodiments, the HIC medium contains sepharose or a derivatizedsepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, orbutyl-s sepharose. In other embodiments, the affinity chromatographicmedium contains material derivatized with mucin type I, II, III, IV, V,or VI, or oligosaccharides derived from or similar to those of mucinstype I, II, III, IV, V, or VI. Alternatively, the affinitychromatographic medium contains material derivatized with antibodiesthat recognize spore-forming bacteria.

Mechanical Treatments.

Provided herein is the physical disruption of the fecal material,particularly by one or more mechanical treatment such as blending,mixing, shaking, vortexing, impact pulverization, and sonication. Asprovided herein, the mechanical disrupting treatment substantiallydisrupts a non-spore material present in the fecal material and does notsubstantially disrupt a spore present in the fecal material, or it maydisrupt the spore material less than the non-spore material, e.g. 2-foldless, 5-, 10-, 30-, 100-, 300-, 1000- or greater than 1000-fold less.Furthermore, mechanical treatment homogenizes the material forsubsequent sampling, testing, and processing. Mechanical treatmentsoptionally include filtration treatments, where the desired sporepopulations are retained on a filter while the undesirable (non-spore)fecal components to pass through, and the spore fraction is thenrecovered from the filter medium. Alternatively, undesirableparticulates and eukaryotic cells may be retained on a filter whilebacterial cells including spores pass through. In some embodiments thespore fraction retained on the filter medium is subjected to adiafiltration step, wherein the retained spores are contacted with awash liquid, typically a sterile saline-containing solution or otherdiluent such as a water compatible polymer including a low-molecularpolyethylene glycol (PEG) solution, in order to further reduce or removethe undesirable fecal components.

Thermal Treatments.

Provided herein is the thermal disruption of the fecal material.Generally, the fecal material is mixed in a saline-containing solutionsuch as phosphate-buffered saline (PBS) and subjected to a heatedenvironment, such as a warm room, incubator, water-bath, or the like,such that efficient heat transfer occurs between the heated environmentand the fecal material. Preferably the fecal material solution is mixedduring the incubation to enhance thermal conductivity and disruptparticulate aggregates. Thermal treatments can be modulated by thetemperature of the environment and/or the duration of the thermaltreatment. For example, the fecal material or a liquid comprising thefecal material is subjected to a heated environment, e.g., a hot waterbath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at leastabout 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more than 10 hours. In certain embodiments the thermal treatmentoccurs at two different temperatures, such as 30 seconds in a 100 degreeCelsius environment followed by 10 minutes in a 50 degree Celsiusenvironment. In preferred embodiments the temperature and duration ofthe thermal treatment are sufficient to kill or remove pathogenicmaterials while not substantially damaging or reducing thegermination-competency of the spores. In other preferred embodiments,the temperature and duration of the thermal treatment is short enough toreduce the germination of the spore population.

Irradiation Treatments.

Provided are methods of treating the fecal material or separatedcontents of the fecal material with ionizing radiation, typically gammairradiation, ultraviolet irradiation or electron beam irradiationprovided at an energy level sufficient to kill pathogenic materialswhile not substantially damaging the desired spore populations. Forexample, ultraviolet radiation at 254 nm provided at an energy levelbelow about 22,000 microwatt seconds per cm² will not generally destroydesired spores.

Centrifugation and Density Separation Treatments.

Provided are methods of separating desired spore populations from theother components of the fecal material by centrifugation. A solutioncontaining the fecal material is subjected to one or more centrifugationtreatments, e.g., at about 200×g, 1000×g, 2000×g, 3000×g, 4000×g,5000×g, 6000×g, 7000×g, 8000×g or greater than 8000×g. Differentialcentrifugation separates desired spores from undesired non-sporematerial; at low forces the spores are retained in solution, while athigher forces the spores are pelleted while smaller impurities (e.g.,virus particles, phage, microscopic fibers, biological macromoleculessuch as free protein, nucleic acids and lipids) are retained insolution. For example, a first low force centrifugation pellets fibrousmaterials; a second, higher force centrifugation pellets undesiredeukaryotic cells, and a third, still higher force centrifugation pelletsthe desired spores while smaller contaminants remain in suspension. Insome embodiments density or mobility gradients or cushions (e.g., stepcushions), such as CsCl, Percoll, Ficoll, Nycodenz, Histodenz or sucrosegradients, are used to separate desired spore populations from othermaterials in the fecal material.

Also provided herein are methods of producing spore populations thatcombine two or more of the treatments described herein in order tosynergistically purify the desired spores while killing or removingundesired materials and/or activities from the spore population. It isgenerally desirable to retain the spore populations undernon-germinating and non-growth promoting conditions and media, in orderto minimize the growth of pathogenic bacteria present in the sporepopulations and to minimize the germination of spores into vegetativebacterial cells.

Purified Spore Populations.

As described herein, purified spore populations contain combinations ofcommensal bacteria of the human gut microbiota with the capacity tomeaningfully provide functions of a healthy microbiota when administeredto a mammalian subject. Without being limited to a specific mechanism,it is thought that such compositions inhibit the growth of a pathogensuch as C. difficile, Salmonella spp., enteropathogenic E. coli,Fusobacterium spp., Klebsiella spp. and vancomycin-resistantEnterococcus spp., so that a healthy, diverse and protective microbiotacan be maintained or, in the case of pathogenic bacterial infectionssuch as C. difficile infection, repopulate the intestinal lumen toreestablish ecological control over potential pathogens. In oneembodiment, the purified spore populations can engraft in the host andremain present for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 10 days, 14 days, 21 days, 25 days, 30 days, 60 days, 90 days, orlonger than 90 days. Additionally, the purified spore populations caninduce other healthy commensal bacteria found in a healthy gut toengraft in the host that are not present in the purified sporepopulations or present at lesser levels and therefore these species areconsidered to “augment” the delivered spore populations. In this manner,commensal species augmentation of the purified spore population in therecipient's gut leads to a more diverse population of gut microbiotathen present initially.

Preferred bacterial genera include Acetanaerobacterium, Acetivibrio,Alicyclobacillus, Alkaliphilus, Anaerofustis, Anaerosporobacter,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Bacteroides,Blautia, Brachyspira, Brevibacillus, Bryantella, Bulleidia,Butyricicoccus, Butyrivibrio, Catenibacterium, Chlamydiales,Clostridiaceae, Clostridiales, Clostridium, Collinsella, Coprobacillus,Coprococcus, Coxiella, Deferribacteres, Desulfitobacterium,Desulfotomaculum, Dorea, Eggerthella, Erysipelothrix,Erysipelotrichaceae, Ethanoligenens, Eubacterium, Faecalibacterium,Filifactor, Flavonifractor, Flexistipes, Fulvimonas, Fusobacterium,Gemmiger, Geobacillus, Gloeobacter, Holdemania,Hydrogenoanaerobacterium, Kocuria, Lachnobacterium, Lachnospira,Lachnospiraceae, Lactobacillus, Lactonifactor, Leptospira, Lutispora,Lysinibacillus, Mollicutes, Moorella, Nocardia, Oscillibacter,Oscillospira, Paenibacillus, Papillibacter, Pseudoflavonifractor,Robinsoniella, Roseburia, Ruminococcaceae, Ruminococcus,Saccharomonospora, Sarcina, Solobacterium, Sporobacter,Sporolactobacillus, Streptomyces, Subdoligranulum, Sutterella,Syntrophococcus, Thermoanaerobacter, Thermobifida, Turicibacter

Preferred bacterial species are provided at Table 1 and demarcated asspore formers. Where specific strains of a species are provided, one ofskill in the art will recognize that other strains of the species can besubstituted for the named strain.

In some embodiments, spore-forming bacteria are identified by thepresence of nucleic acid sequences that modulate sporulation. Inparticular, signature sporulation genes are highly conserved acrossmembers of distantly related genera including Clostridium and Bacillus.Traditional approaches of forward genetics have identified many, if notall, genes that are essential for sporulation (spo). The developmentalprogram of sporulation is governed in part by the successive action offour compartment-specific sigma factors (appearing in the order σf, σE,σG and σK), whose activities are confined to the forespore (σF and σG)or the mother cell (σE and σK). In other embodiments, spore-formingbacteria are identified by the biochemical activity of DPA producingenzymes or by analyzing DPA content of cultures. As part of thebacterial sporulation, large amounts of DPA are produced, and comprise5-15% of the mass of a spore. Because not all viable spores germinateand grow under known media conditions, it is difficult to assess a totalspore count in a population of bacteria. As such, a measurement of DPAcontent highly correlates with spore content and is an appropriatemeasure for characterizing total spore content in a bacterialpopulation.

[Provided are spore populations containing more than one type ofbacterium. As used herein, a “type” or more than one “types” of bacteriamay be differentiated at the genus level, the species, level, thesub-species level, the strain level or by any other taxonomic method, asdescribed herein and otherwise known in the art.

In some embodiments all or essentially all of the bacterial sporespresent in a purified population are obtained from a fecal materialtreated as described herein or otherwise known in the art. Inalternative embodiments, one or more than one bacterial spores or typesof bacterial spores are generated in culture and combined to form apurified spore population. In other alternative embodiments, one or moreof these culture-generated spore populations are combined with a fecalmaterial-derived spore population to generate a hybrid spore population.Bacterial compositions may contain at least two types of these preferredbacteria, including strains of the same species. For instance, abacterial composition may comprise at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or at least 20 or morethan 20 types of bacteria, as defined by species or operationaltaxonomic unit (OTU) encompassing such species.

Thus, provided herein are methods for production of a compositioncontaining a population of bacterial spores suitable for therapeuticadministration to a mammalian subject in need thereof. And thecomposition is produced by generally following the steps of: (a)providing a fecal material obtained from a mammalian donor subject; and(b) subjecting the fecal material to at least one purification treatmentor step under conditions such that a population of bacterial spores isproduced from the fecal material. The composition is formulated suchthat a single oral dose contains at least about 1×10⁴ colony formingunits of the bacterial spores, and a single oral dose will typicallycontain about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ CFUs of thebacterial spores. The presence and/or concentration of a given type ofbacterial spore may be known or unknown in a given purified sporepopulation. If known, for example the concentration of spores of a givenstrain, or the aggregate of all strains, is e.g., 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, orgreater than 1×10¹⁵ viable bacterial spores per gram of composition orper administered dose.

In some formulations, the composition contains at least about 0.5%, 1%,2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90%spores on a mass basis. In some formulations, the administered dose doesnot exceed 200, 300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.

The bacterial spore compositions are generally formulated for oral orgastric administration, typically to a mammalian subject. In particularembodiments, the composition is formulated for oral administration as asolid, semi-solid, gel, or liquid form, such as in the form of a pill,tablet, capsule, or lozenge. In some embodiments, such formulationscontain or are coated by an enteric coating to protect the bacteriathrough the stomach and small intestine, although spores are generallyresistant to the stomach and small intestines. In other embodiments, thebacterial spore compositions may be formulated with a germinant toenhance engraftment, or efficacy. In yet other embodiments, thebacterial spore compositions may be co-formulated or co-administeredwith prebiotic substances, to enhance engraftment or efficacy.

The bacterial spore compositions may be formulated to be effective in agiven mammalian subject in a single administration or over multipleadministrations. For example, a single administration is substantiallyeffective to reduce Cl. difficile and/or Cl. difficile toxin content ina mammalian subject to whom the composition is administered.Substantially effective means that Cl. difficile and/or Cl. difficiletoxin content in the subject is reduced by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% followingadministration of the composition. Alternatively, efficacy may bemeasured by the absence of diarrheal symptoms or the absence of carriageof C. difficile or C. difficile toxin after 2 day, 4 days, 1 week, 2weeks, 4 weeks, 8 weeks or longer than 8 weeks.

Bacterial Compositions

Provided are bacteria and combinations of bacteria of the human gutmicrobiota with the capacity to meaningfully provide functions of ahealthy microbiota when administered to mammalian hosts. Without beinglimited to a specific mechanism, it is thought that such compositionsinhibit the growth, proliferation, and/or colonization of one or aplurality of pathogenic bacteria in the dysbiotic microbiotal niche, sothat a healthy, diverse and protective microbiota colonizes andpopulates the intestinal lumen to establish or reestablish ecologicalcontrol over pathogens or potential pathogens (e.g., some bacteria arepathogenic bacteria only when present in a dysbiotic environment).Inhibition of pathogens includes those pathogens such as C. difficile,Salmonella spp., enteropathogenic E coli, multi-drug resistant bacteriasuch as Klebsiella, and E. coli, Carbapenem-resistent Enterobacteriaceae(CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), andvancomycin-resistant Enterococci (VRE).

As used herein, a “type” or more than one “types” of bacteria may bedifferentiated at the genus level, the species, level, the sub-specieslevel, the strain level or by any other taxonomic method, as describedherein and otherwise known in the art.

Bacterial compositions may comprise two types of bacteria (termed“binary combinations” or “binary pairs”) or greater than two types ofbacteria. For instance, a bacterial composition may comprise at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, or at least 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or at least 40, at least 50 or greater than 50types of bacteria, as defined by species or operational taxonomic unit(OTU), or otherwise as provided herein.

In another embodiment, the number of types of bacteria present in abacterial composition is at or below a known value. For example, in suchembodiments the bacterial composition comprises 50 or fewer types ofbacteria, such as 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, or 10 or fewer, or 9 or fewer types ofbacteria, 8 or fewer types of bacteria, 7 or fewer types of bacteria, 6or fewer types of bacteria, 5 or fewer types of bacteria, 4 or fewertypes of bacteria, or 3 or fewer types of bacteria. In anotherembodiment, a bacterial composition comprises from 2 to no more than 40,from 2 to no more than 30, from 2 to no more than 20, from 2 to no morethan 15, from 2 to no more than 10, or from 2 to no more than 5 types ofbacteria.

Bacterial Compositions Described by Species

Bacterial compositions may be prepared comprising at least two types ofisolated bacteria, chosen from the species in Table 1.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Enterococcus faecalis(previously known as Streptococcus faecalis), Clostridium innocuum,Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus,Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1),Clostridum bifermentans, and Blautia producta (previously known asPeptostreptococcus productus). In an alternative embodiment, at leastone of the preceding species is not substantially present in thebacterial composition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Enterococcus faecalis(previously known as Streptococcus faecalis), Clostridium innocuum,Clostridium ramosum, Bacteroides ovatus, Bacteroides vulgatus,Bacteroides thetaoiotaomicron, Escherichia coli (1109 and 1108-1),Clostridum bifermentans, and Blautia producta (previously known asPeptostreptococcus productus). In an alternative embodiment, at leastone of the preceding species is not substantially present in thebacterial composition.

In another embodiment, the bacterial composition comprises at least oneand preferably more than one of the following: Acidaminococcusintestinalis, Bacteroides ovatus, two strains of Bifidobacteriumadolescentis, two strains of Bifidobacterium longum, Blautia producta,Clostridium cocleatum, Collinsella aerofaciens, two strains of Dorealongicatena, Escherichia coli, Eubacterium desmolans, Eubacteriumeligens, Eubacterium limosum, four strains of Eubacterium rectale,Eubacterium ventriosumi, Faecalibacterium prausnitzii, Lachnospirapectinoshiza, Lactobacillus casei, Lactobacillus casei/paracasei,Paracateroides distasonis, Raoultella sp., one strain of Roseburia(chosen from Roseburia faecalis or Roseburia faecis), Roseburiaintestinalis, two strains of Ruminococcus torques, two strains ofRuminococcus obeum, and Streptococcus mitis. In an alternativeembodiment, at least one of the preceding species is not substantiallypresent in the bacterial composition.

In yet another embodiment, the bacterial composition comprises at leastone and preferably more than one of the following: Barnesiellaintestinihominis; Lactobacillus reuteri; a species characterized as oneof Enterococcus hirae, Enterococus faecium, or Enterococcus durans; aspecies characterized as one of Anaerostipes caccae or Clostridiumindolis; a species characterized as one of Staphylococcus warneri orStaphylococcus pasteuri; and Adlercreutzia equolifaciens. In analternative embodiment, at least one of the preceding species is notsubstantially present in the bacterial composition.

In other embodiments, the bacterial composition comprises at least oneand preferably more than one of the following: Clostridium absonum,Clostridium argentinense, Clostridium baratii, Clostridium bartlettii,Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum,Clostridium cadaveris, Clostridium camis, Clostridium celatum,Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium difficile, Clostridium fallax, Clostridiumfelsineum, Clostridium ghonii, Clostridium glycolicum, Clostridiumhaemolyticum, Clostridium hastiforme, Clostridium histolyticum,Clostridium indolis, Clostridium innocuum, Clostridium irregulare,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense,Clostridium sartagoforme, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum,Clostridium tertium, Clostridium tetani, Clostridium welchii, andClostridium villosum. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Clostridium innocuum,Clostridum bifermentans, Clostridium butyricum, Bacteroides fragilis,Bacteroides thetaiotaomicron, Bacteroides uniformis, three strains ofEscherichia coli, and Lactobacillus sp. In an alternative embodiment, atleast one of the preceding species is not substantially present in thebacterial composition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Clostridium bifermentans,Clostridium innocuum, Clostridium butyricum, three strains ofEscherichia coli, three strains of Bacteroides, and Blautia producta. Inan alternative embodiment, at least one of the preceding species is notsubstantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Bacteroides sp., Escherichiacoli, and non pathogenic Clostridia, including Clostridium innocuum,Clostridium bifermentans and Clostridium ramosum. In an alternativeembodiment, at least one of the preceding species is not substantiallypresent in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Bacteroides species,Escherichia coli and non-pathogenic Clostridia, such as Clostridiumbutyricum, Clostridium bifermentans and Clostridium innocuum. In analternative embodiment, at least one of the preceding species is notsubstantially present in the bacterial composition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Bacteroides caccae,Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis,Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis,Bacteroides fragilis-ryhm, Bacteroides gracilis, Bacteroides levii,Bacteroides macacae, Bacteroides merdae, Bacteroides ovatus, Bacteroidespneumosintes, Bacteroides putredinis, Bacteroides pyogenes, Bacteroidessplanchnicus, Bacteroides stercoris, Bacteroides tectum, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, andBacteroides vulgatus. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Bacteroides, Eubacteria,Fusobacteria, Propionibacteria, Lactobacilli, anaerobic cocci,Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas, andPeptostreptococcus. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

In one embodiment, the bacterial composition comprises at least one andpreferably more than one of the following: Bacteroides fragilis ss.Vulgatus, Eubacterium aerofaciens, Bacteroides fragilis ss.Thetaiotaomicron, Blautia producta (previously known asPeptostreptococcus productus II), Bacteroides fragilis ss. Distasonis,Fusobacterium prausnitzii, Coprococcus eutactus, Eubacterium aerofaciensIII, Blautia producta (previously known as Peptostreptococcus productusI), Ruminococcus bronii, Bifidobacterium adolescentis, Gemmigerformicilis, Bifidobacterium longum, Eubacterium siraeum, Ruminococcustorques, Eubacterium rectale Eubacterium rectale IV, Eubacteriumeligens, Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilisss. A, Eubacterium biforme, Bifidobacterium infantis, Eubacteriumrectale Coprococcus comes, Bacteroides capillosus, Ruminococcus albus,Eubacterium formicigenerans, Eubacterium hallii, Eubacterium ventriosumI, Fusobacterium russii, Ruminococcus obeum, Eubacterium rectale II,Clostridium ramosum I, Lactobacillus leichmanii, Ruminococcus cailidus,Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacteriumventriosum, Bacteroides fragilis ss. fragilis, Bacteroides AR,Coprococcus catus, Eubacterium hadrum, Eubacterium cylindroides,Eubacterium ruminantium, Eubacterium CH-1, Staphylococcus epidermidis,Peptostreptococcus BL, Eubacterium limosum, Bacteroides praeacutus,Bacteroides L, Fusobacterium mortiferum I, Fusobacterium naviforme,Clostridium innocuum, Clostridium ramosum, Propionibacterium acnes,Ruminococcus flavefaciens, Ruminococcus AT, Peptococcus AU-1,Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroides fragilis ss. ovatus,-ss. d, -ss. f, Bacteroides L-1, L-5; Fusobacterium nucleatum,Fusobacterium mortiferum, Escherichia coli, Streptococcus morbiliorum,Peptococcus magnus, Peptococcus G, AU-2; Streptococcus intermedius,Ruminococcus lactaris, Ruminococcus CO Gemmiger X, Coprococcus BH, -CC;Eubacterium tenue, Eubacterium ramulus, Eubacterium AE, -AG-H, -AG-M,-AJ, -BN-1; Bacteroides clostridiiformis ss. clostridliformis,Bacteroides coagulans, Bacteroides orails, Bacteroides ruminicola ss.brevis, -ss. ruminicola, Bacteroides splanchnicus, Desuifomonas pigra,Bacteroides L-4, -N-i; Fusobacterium H, Lactobacillus G, andSuccinivibrio A. In an alternative embodiment, at least one of thepreceding species is not substantially present in the bacterialcomposition.

Bacterial Compositions Described by Operational Taxonomic Unit (OTUs)

Bacterial compositions may be prepared comprising at least two types ofisolated bacteria, chosen from the species in Table 1.

In one embodiment, the OTUs can be characterized by one or more of thevariable regions of the 16S sequence (V1-V9). These regions in bacteriaare defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879,986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. (See, e.g.,Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA genefrom Escherichia coli, PNAS 75(10):4801-4805 (1978)). In someembodiments, at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9regions are used to characterize an OTU. In one embodiment, the V1, V2,and V3 regions are used to characterize an OTU. In another embodiment,the V3, V4, and V5 regions are used to characterize an OTU. In anotherembodiment, the V4 region is used to characterize an OTU.

Bacterial Compositions Exclusive of Certain Bacterial Species or Strains

In one embodiment, the bacterial composition does not comprise at leastone of Enterococcus faecalis (previously known as Streptococcusfaecalis), Clostridium innocuum, Clostridium ramosum, Bacteroidesovatus, Bacteroides vulgatus, Bacteroides thetaoiotaomicron, Escherichiacoli (1109 and 1108-1), Clostridum bifermentans, and Blautia producta(previously known as Peptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise atleast one of Acidaminococcus intestinalis, Bacteroides ovatus, twospecies of Bifidobacterium adolescentis, two species of Bifidobacteriumlongum, Collinsella aerofaciens, two species of Dorea longicatena,Escherichia coli, Eubacterium eligens, Eubacterium limosum, four speciesof Eubacterium rectale, Eubacterium ventriosumi, Faecalibacteriumprausnitzii, Lactobacillus casei, Lactobacillus paracasei,Paracateroides distasonis, Raoultella sp., one species of Roseburia(chosen from Roseburia faecalis or Roseburia faecis), Roseburiaintestinalis, two species of Ruminococcus torques, and Streptococcusmitis.

In yet another embodiment, the bacterial composition does not compriseat least one of Bamesiella intestinihominis; Lactobacillus reuteri; aspecies characterized as one of Enterococcus hirae, Enterococus faecium,or Enterococcus durans; a species characterized as one of Anaerostipescaccae or Clostridium indolis; a species characterized as one ofStaphylococcus warneri or Staphylococcus pasteuri; and Adlercreutziaequolifaciens.

In other embodiments, the bacterial composition does not comprise atleast one of Clostridium absonum, Clostridium argentinense, Clostridiumbaratii, Clostridium bifermentans, Clostridium botulinum, Clostridiumbutyricum, Clostridium cadaveris, Clostridium camis, Clostridiumcelatum, Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium difficile, Clostridium fallax, Clostridiumfelsineum, Clostridium ghonii, Clostridium glycolicum, Clostridiumhaemolyticum, Clostridium hastiforme, Clostridium histolyticum,Clostridium indolis, Clostridium innocuum, Clostridium irregulars,Clostridium limosum, Clostridium malenominatum, Clostridium novyi,Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium ramosum, Clostridium sardiniense,Clostridium sartagoforme, Clostridium scindens, Clostridium septicum,Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,Clostridium sporogenes, Clostridium subterminale, Clostridium symbiosum,Clostridium tertium, Clostridium tetani, Clostridium welchii, andClostridium villosum.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium innocuum, Clostridum bifermentans, Clostridiumbutyricum, Bacteroides fragilis, Bacteroides thetaiotaomicron,Bacteroides uniformis, three strains of Escherichia coli, andLactobacillus sp.

In another embodiment, the bacterial composition does not comprise atleast one of Clostridium bifermentans, Clostridium innocuum, Clostridiumbutyricum, three strains of Escherichia coli, three strains ofBacteroides, and Blautia producta (previously known asPeptostreptococcus productus).

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides sp., Escherichia coli, and non pathogenicClostridia, including Clostridium innocuum, Clostridium bifermentans andClostridium ramosum.

In another embodiment, the bacterial composition does not comprise atleast one of more than one Bacteroides species, Escherichia coli andnon-pathogenic Clostridia, such as Clostridium butyricum, Clostridiumbifermentans and Clostridium innocuum.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides caccae, Bacteroides capillosus, Bacteroidescoagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroidesforsythus, Bacteroides fragilis, Bacteroides fragilis-ryhm, Bacteroidesgracilis, Bacteroides levii, Bacteroides macacae, Bacteroides merdae,Bacteroides ovatus, Bacteroides pneumosintes, Bacteroides putredinis,Bacteroides pyogenes, Bacteroides splanchnicus, Bacteroides stercoris,Bacteroides tectum, Bacteroides thetaiotaomicron, Bacteroides uniformis,Bacteroides ureolyticus, and Bacteroides vulgatus.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides, Eubacteria, Fusobacteria, Propionibacteria,Lactobacilli, anaerobic cocci, Ruminococcus, Escherichia coli, Gemmiger,Desulfomonas, and Peptostreptococcus.

In another embodiment, the bacterial composition does not comprise atleast one of Bacteroides fragilis ss. Vulgatus, Eubacterium aerofaciens,Bacteroides fragilis ss. Thetaiotaomicron, Blautia producta (previouslyknown as Peptostreptococcus productus II), Bacteroides fragilis ss.Distasonis, Fusobacterium prausnitzii, Coprococcus eutactus, Eubacteriumaerofaciens III, Blautia producta (previously known asPeptostreptococcus productus I), Ruminococcus bromii, Bifidobacteriumadolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacteriumsiraeum, Ruminococcus torques, Eubacterium rectale III-H, Eubacteriumrectale IV, Eubacterium eligens, Bacteroides eggerthii, Clostridiumleptum, Bacteroides fragilis ss. A, Eubacterium biforme, Bifidobacteriuminfantis, Eubacterium rectale III-F, Coprococcus comes, Bacteroidescapillosus, Ruminococcus albus, Eubacterium formicigenerans, Eubacteriumhallii, Eubacterium ventriosum I, Fusobacterium russii, Ruminococcusobeum, Eubacterium rectale II, Clostridium ramosum I, Lactobacillusleichmanii, Ruminococcus cailidus, Butyrivibrio crossotus,Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilisss. fragilis, Bacteroides AR, Coprococcus catus, Eubacterium hadrum,Eubacterium cylindroides, Eubacterium ruminantium, Eubacterium CH-1,Staphylococcus epidermidis, Peptostreptococcus BL, Eubacterium limosum,Bacteroides praeacutus, Bacteroides L, Fusobacterium mortiferum I,Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum,Propionibacterium acnes, Ruminococcus flavefaciens, Ruminococcus AT,Peptococcus AU-1, Eubacterium AG, -AK, -AL, -AL-1, -AN; Bacteroidesfragilis ss. ovatus, -ss. d, -ss. f; Bacteroides L-1, L-5; Fusobacteriumnucleatum, Fusobacterium mortiferum, Escherichia coli, Streptococcusmorbiliorum, Peptococcus magnus, Peptococcus G, AU-2; Streptococcusintermedius, Ruminococcus lactaris, Ruminococcus CO Gemmiger X,Coprococcus BH, -CC, Eubacterium tenue, Eubacterium ramulus, EubacteriumAE, -AG-H, -AG-M, -AJ, -BN-1, Bacteroides clostridiiformis ss.clostridliformis, Bacteroides coagulans, Bacteroides orails, Bacteroidesruminicola ss. brevis, -ss. ruminicola, Bacteroides splanchnicus,Desuifomonas pigra, Bacteroides L-4, -N-i; Fusobacterium H,Lactobacillus G, and Succinivibrio A.

Inhibition of Bacterial Pathogens

In some embodiments, the bacterial composition provides a protective ortherapeutic effect against infection by one or more GI pathogens ofinterest.

A list of exemplary bacterial pathogens is provided in Table 1 asindicated by pathogen status.

In some embodiments, the pathogenic bacterium is selected from the groupconsisting of Yersinia, Vibrio, Treponema, Streptococcus,Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas,Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella,Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia,Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium,Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella,Borrelia, Bordetella, Bifidobacterium, Bacillus, multi-drug resistantbacteria, extended spectrum beta-lactam resistant Enterococci (ESBL),Carbapenem-resistent Enterobacteriaceae (CRE), and vancomycin-resistantEnterococci (VRE).

In some embodiments, these pathogens include, but are not limited to,Aeromonas hydrophila, Campylobacter fetus, Plesiomonas shigelloides,Bacillus cereus, Campylobacter jejuni, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, enteroaggregativeEscherichia coli, enterohemorrhagic Escherichia coli, enteroinvasiveEscherichia coli, enterotoxigenic Escherichia coli (such as, but notlimited to, LT and/or ST), Escherichia coli 0157:H7, Helicobacterpylori, Klebsiellia pneumonia, Lysteria monocytogenes, Plesiomonasshigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi,Shigella spp., Staphylococcus spp., Staphylococcus aureus,vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae,Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica.

In one embodiment, the pathogen of interest is at least one pathogenchosen from Clostridium difficile, Salmonella spp., pathogenicEscherichia coli, vancomycin-resistant Enterococcus spp., and extendedspectrum beta-lactam resistant Enterococci (ESBL).

Purified Spore Populations

In some embodiments, the bacterial compositions comprise purified sporepopulations. Purified spore populations contain combinations ofcommensal bacteria of the human gut microbiota with the capacity tomeaningfully provide functions of a healthy microbiota when administeredto a mammalian subject. Without being limited to a specific mechanism,it is thought that such compositions inhibit the growth of a pathogensuch as C. difficile, Salmonella spp., enteropathogenic E. coli, andvancomycin-resistant Enterococcus spp., so that a healthy, diverse andprotective microbiota can be maintained or, in the case of pathogenicbacterial infections such as C. difficile infection, repopulate theintestinal lumen to reestablish ecological control over potentialpathogens. In some embodiments, yeast spores and other fungal spores arealso purified and selected for therapeutic use.

Disclosed herein are therapeutic compositions containing non-pathogenic,germination-competent bacterial spores, for the prevention, control, andtreatment of gastrointestinal diseases, disorders and conditions and forgeneral nutritional health. These compositions are advantageous in beingsuitable for safe administration to humans and other mammalian subjectsand are efficacious in numerous gastrointestinal diseases, disorders andconditions and in general nutritional health. While spore-basedcompositions are known, these are generally prepared according tovarious techniques such as lyophilization or spray-drying of liquidbacterial cultures, resulting in poor efficacy, instability, substantialvariability and lack of adequate safety.

It has now been found that populations of bacterial spores can beobtained from biological materials obtained from mammalian subjects,including humans. These populations are formulated into compositions asprovided herein, and administered to mammalian subjects using themethods as provided herein.

Provided herein are therapeutic compositions containing a purifiedpopulation of bacterial spores. As used herein, the terms “purify”,“purified” and “purifying” refer to the state of a population (e.g., aplurality of known or unknown amount and/or concentration) of desiredbacterial spores, that have undergone one or more processes ofpurification, e.g., a selection or an enrichment of the desiredbacterial spore, or alternatively a removal or reduction of residualhabitat products as described herein. In some embodiments, a purifiedpopulation has no detectable undesired activity or, alternatively, thelevel or amount of the undesired activity is at or below an acceptablelevel or amount. In other embodiments, a purified population has anamount and/or concentration of desired bacterial spores at or above anacceptable amount and/or concentration. In other embodiments, thepurified population of bacterial spores is enriched as compared to thestarting material (e.g., a fecal material) from which the population isobtained. This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, orgreater than 99.9999% as compared to the starting material.

In certain embodiments, the purified populations of bacterial sporeshave reduced or undetectable levels of one or more pathogenicactivities, such as toxicity, an infection of the mammalian recipientsubject, an immunomodulatory activity, an autoimmune response, ametabolic response, or an inflammatory response or a neurologicalresponse. Such a reduction in a pathogenic activity may be by 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to thestarting material. In other embodiments, the purified populations ofbacterial spores have reduced sensory components as compared to fecalmaterial, such as reduced odor, taste, appearance, and umami.

Provided are purified populations of bacterial spores that aresubstantially free of residual habitat products. In certain embodiments,this means that the bacterial spore composition no longer contains asubstantial amount of the biological matter associated with themicrobial community while living on or in the human or animal subject,and the purified population of spores may be 100% free, 99% free, 98%free, 97% free, 96% free, or 95% free of any contamination of thebiological matter associated with the microbial community. Substantiallyfree of residual habitat products may also mean that the bacterial sporecomposition contains no detectable cells from a human or animal, andthat only microbial cells are detectable, in particular, only desiredmicrobial cells are detectable. In another embodiment, it means thatfewer than 1×10⁻²%, 1×10⁻³%, 1×10⁻⁴%, 1×10⁻⁵%, 1×10⁻⁶%, 1×10⁻⁷%, 1×10⁻⁸%of the cells in the bacterial composition are human or animal, ascompared to microbial cells. In another embodiment, the residual habitatproduct present in the purified population is reduced at least a certainlevel from the fecal material obtained from the mammalian donor subject,e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, orgreater than 99.9999%.

In one embodiment, substantially free of residual habitat products orsubstantially free of a detectable level of a pathogenic material meansthat the bacterial composition contains no detectable viral (includingbacterial viruses (i.e., phage)), fungal, or mycoplasmal or toxoplasmalcontaminants, or a eukaryotic parasite such as a helminth.Alternatively, the purified spore populations are substantially free ofan acellular material, e.g., DNA, viral coat material, or non-viablebacterial material.

As described herein, purified spore populations can be demonstrated bygenetic analysis (e.g., PCR, DNA sequencing), serology and antigenanalysis, and methods using instrumentation such as flow cytometry withreagents that distinguish desired bacterial spores from non-desired,contaminating materials.

Exemplary biological materials include fecal materials such as feces ormaterials isolated from the various segments of the small and largeintestines. Fecal materials are obtained from a mammalian donor subject,or can be obtained from more than one donor subject, e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400,500, 750, 1000 or from greater than 1000 donors, where such materialsare then pooled prior to purification of the desired bacterial spores.

In alternative embodiments, the desired bacterial spores are purifiedfrom a single fecal material sample obtained from a single donor, andafter such purification are combined with purified spore populationsfrom other purifications, either from the same donor at a differenttime, or from one or more different donors, or both.

Preferred bacterial genera include Acetonema, Alkaliphilus,Alicyclobacillus, Amphibacillus, Ammonifex, Anaerobacter, Anaerofustis,Anaerostipes, Anaerotruncus, Anoxybacillus, Bacillus, Blautia,Brevibacillus, Bryantella, Caldicellulosiruptor, Caloramator,Candidatus, Carboxydibrachium, Carboxydothermus, Clostridium, Cohnella,Coprococcus, Dendrosporobacter Desulfitobacterium, Desulfosporosinus,Desulfotomaculum, Dorea, Eubacterium, Faecalibacterium, Filifactor,Geobacillus, Halobacteroides, Heliobacillus, Heliobacterium,Heliophilum, Heliorestis, Lachnoanaerobaculum, Lysinibacillus, Moorella,Oceanobacillus, Orenia (S.), Oxalophagus, Oxobacter, Paenibacillus,Pelospora, Pelotomaculum, Propionispora, Roseburia, Ruminococcus,Sarcina, Sporobacterium, Sporohalobacter, Sporolactobacillus, Sporomusa,Sporosarcina, Sporotomaculum, Subdoligranulum, Symbiobacterium,Syntrophobotulus, Syntrophospora, Terribacillus, Thermoanaerobacter, andThermosinus.

Preferred bacterial species are provided at Table X4. Where specificstrains of a species are provided, one of skill in the art willrecognize that other strains of the species can be substituted for thenamed strain.

In some embodiments, spore-forming bacteria are identified by thepresence of nucleic acid sequences that modulate sporulation. Inparticular, signature sporulation genes are highly conserved acrossmembers of distantly related genera including Clostridium and Bacillus.Traditional approaches of forward genetics have identified many, if notall, genes that are essential for sporulation (spo). The developmentalprogram of sporulation is governed in part by the successive action offour compartment-specific sigma factors (appearing in the order σf, σE,σG and σK), whose activities are confined to the forespore (σF and σG)or the mother cell (σE and σK).

Provided are spore populations containing more than one type ofbacterium. As used herein, a “type” or more than one “types” of bacteriamay be differentiated at the genus level, the species, level, thesub-species level, the strain level or by any other taxonomic method, asdescribed herein and otherwise known in the art.

In some embodiments, all or essentially all of the bacterial sporespresent in a purified population are obtained from a fecal materialtreated as described herein or otherwise known in the art. Inalternative embodiments, one or more than one bacterial spores or typesof bacterial spores are generated in culture and combined to form apurified spore population. In other alternative embodiments, one or moreof these culture-generated spore populations are combined with a fecalmaterial-derived spore population to generate a hybrid spore population.Bacterial compositions may contain at least two types of these preferredbacteria, including strains of the same species. For instance, abacterial composition may comprise at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, or at least 20 or morethan 20 types of bacteria, as defined by species or operationaltaxonomic unit (OTU) encompassing such species.

Thus, provided herein are methods for production of a compositioncontaining a population of bacterial spores suitable for therapeuticadministration to a mammalian subject in need thereof. And thecomposition is produced by generally following the steps of: (a)providing a fecal material obtained from a mammalian donor subject; and(b) subjecting the fecal material to at least one purification treatmentor step under conditions such that a population of bacterial spores isproduced from the fecal material. The composition is formulated suchthat a single oral dose contains at least about 1×10⁴ colony formingunits of the bacterial spores, and a single oral dose will typicallycontain about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, or greater than 1×10¹⁵ CFUs of thebacterial spores. The presence and/or concentration of a given type ofbacteria spore may be known or unknown in a given purified sporepopulation. If known, for example the concentration of spores of a givenstrain, or the aggregate of all strains, is e.g., 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, orgreater than 1×10¹⁵ viable bacterial spores per gram of composition orper administered dose.

In some formulations, the composition contains at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than 90% spores on a massbasis. In some formulations, the administered dose does not exceed 200,300, 400, 500, 600, 700, 800, 900 milligrams or 1, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, or 1.9 grams in mass.

The bacterial spore compositions are generally formulated for oral orgastric administration, typically to a mammalian subject. In particularembodiments, the composition is formulated for oral administration as asolid, semi-solid, gel, or liquid form, such as in the form of a pill,tablet, capsule, or lozenge. In some embodiments, such formulationscontain or are coated by an enteric coating to protect the bacteriathrough the stomach and small intestine, although spores are generallyresistant to the stomach and small intestines.

The bacterial spore compositions may be formulated to be effective in agiven mammalian subject in a single administration or over multipleadministrations. For example, a single administration is substantiallyeffective to reduce Cl. difficile and/or Cl. difficile toxin content ina mammalian subject to whom the composition is administered.Substantially effective means that Cl. difficile and/or Cl. difficiletoxin content in the subject is reduced by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater than 99% followingadministration of the composition.

Methods of the Invention

Methods for Determining 16S Sequences

OTUs can be defined either by full 16S sequencing of the rRNA gene, bysequencing of a specific hypervariable region of this gene (i.e. V1, V2,V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination ofhypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial16S rDNA is approximately 1500 nucleotides in length and is used inreconstructing the evolutionary relationships and sequence similarity ofone bacterial isolate to another using phylogenetic approaches. 16Ssequences are used for phylogenetic reconstruction as they are ingeneral highly conserved, but contain specific hypervariable regionsthat harbor sufficient nucleotide diversity to differentiate genera andspecies of most microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S sequence, genomicDNA is extracted from a bacterial sample, the 16S rDNA (full region orspecific hypervariable regions) amplified using polymerase chainreaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingcan be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

OTUs can be defined by a combination of nucleotide markers or genes, inparticular highly conserved genes (e.g., “house-keeping” genes), or acombination thereof, full-genome sequence, or partial genome sequencegenerated using amplified genetic products, or whole genome sequence(WGS). Using well defined methods DNA extracted from a bacterial samplewill have specific genomic regions amplified using PCR and sequenced todetermine the nucleotide sequence of the amplified products. In thewhole genome shotgun (WGS) method, extracted DNA will be directlysequenced without amplification. Sequence data can be generated usingany sequencing technology including, but not limited to Sanger,Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences,and/or Oxford Nanopore.

Methods for Preparing a Bacterial Composition for Administration to aSubject

Methods for producing bacterial compositions can include three mainprocessing steps, combined with one or more mixing steps. The stepsinclude organism banking, organism production, and preservation.

For banking, the strains included in the bacterial composition may be(1) isolated directly from a specimen or taken from a banked stock, (2)optionally cultured on a nutrient agar or broth that supports growth togenerate viable biomass, and (3) the biomass optionally preserved inmultiple aliquots in long-term storage.

In embodiments that use a culturing step, the agar or broth can containnutrients that provide essential elements and specific factors thatenable growth. An example would be a medium composed of 20 g/L glucose,10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/Lsodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/Lmagnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1mg/L menadione. A variety of microbiological media and variations arewell known in the art (e.g. R. M. Atlas, Handbook of MicrobiologicalMedia (2010) CRC Press). Medium can be added to the culture at thestart, may be added during the culture, or may beintermittently/continuously flowed through the culture. The strains inthe bacterial composition may be cultivated alone, as a subset of thebacterial composition, or as an entire collection comprising thebacterial composition. As an example, a first strain may be cultivatedtogether with a second strain in a mixed continuous culture, at adilution rate lower than the maximum growth rate of either cell toprevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for atime sufficient to build biomass. For bacterial compositions for humanuse, this is often at 37° C. temperature, pH, and other parameter withvalues similar to the normal human niche. The environment can beactively controlled, passively controlled (e.g., via buffers), orallowed to drift. For example, for anaerobic bacterial compositions(e.g., gut microbiota), an anoxic/reducing environment can be employed.This can be accomplished by addition of reducing agents such as cysteineto the broth, and/or stripping it of oxygen. As an example, a culture ofa bacterial composition can be grown at 37° C., pH 7, in the mediumabove, pre-reduced with 1 g/L cysteine HCl.

When the culture has generated sufficient biomass, it can be preservedfor banking. The organisms can be placed into a chemical milieu thatprotects from freezing (adding tryoprotectants′), drying(‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensinginto multiple (optionally identical) containers to create a uniformbank, and then treating the culture for preservation. Containers aregenerally impermeable and have closures that assure isolation from theenvironment. Cryopreservation treatment is accomplished by freezing aliquid at ultra-low temperatures (e.g., at or below −80° C.). Driedpreservation removes water from the culture by evaporation (in the caseof spray drying or ‘cool drying’) or by sublimation (e.g., for freezedrying, spray freeze drying). Removal of water improves long-termbacterial composition storage stability at temperatures elevated abovecryogenic. If the bacterial composition comprises spore forming speciesand results in the production of spores, the final composition can bepurified by additional means, such as density gradient centrifugationpreserved using the techniques described above. Bacterial compositionbanking can be done by culturing and preserving the strainsindividually, or by mixing the strains together to create a combinedbank. As an example of cryopreservation, a bacterial composition culturecan be harvested by centrifugation to pellet the cells from the culturemedium, the supernate decanted and replaced with fresh culture brothcontaining 15% glycerol. The culture can then be aliquoted into 1 mLcryotubes, sealed, and placed at −80° C. for long-term viabilityretention. This procedure achieves acceptable viability upon recoveryfrom frozen storage.

Organism production can be conducted using similar culture steps tobanking, including medium composition and culture conditions. It can beconducted at larger scales of operation, especially for clinicaldevelopment or commercial production. At larger scales, there can beseveral subcultivations of the bacterial composition prior to the finalcultivation. At the end of cultivation, the culture is harvested toenable further formulation into a dosage form for administration. Thiscan involve concentration, removal of undesirable medium components,and/or introduction into a chemical milieu that preserves the bacterialcomposition and renders it acceptable for administration via the chosenroute. For example, a bacterial composition can be cultivated to aconcentration of 10¹⁰ CFU/mL, then concentrated 20-fold by tangentialflow microfiltration; the spent medium can be exchanged by diafilteringwith a preservative medium consisting of 2% gelatin, 100 mM trehalose,and 10 mM sodium phosphate buffer. The suspension can then befreeze-dried to a powder and titrated.

After drying, the powder can be blended to an appropriate potency, andmixed with other cultures and/or a filler such as microcrystallinecellulose for consistency and ease of handling, and the bacterialcomposition formulated as provided herein.

Administration of Bacterial Compositions.

The bacterial compositions of the invention are suitable foradministration to mammals and non-mammalian animals in need thereof. Incertain embodiments, the mammalian subject is a human subject who hasone or more symptoms of a dysbiosis, including but not limited toovergrowth of an undesired pathobiont or pathogen, reducedrepresentation of key bacterial taxa such as the Bacteroidetes orFirmicutes or genera or species thereof, or reduced diversity ofmicrobial species compared to a healthy individual, or reduced overallabundance of anaerobic bacteria.

When the mammalian subject is suffering from a disease, disorder orcondition characterized by an aberrant microbiota, the bacterialcompositions described herein are suitable for treatment thereof. Insome embodiments, the mammalian subject has not received antibiotics inadvance of treatment with the bacterial compositions. For example, themammalian subject has not been administered at least two doses ofvancomycin, metronidazole and/or or similar antibiotic compound withinone week prior to administration of the therapeutic composition. Inother embodiments, the mammalian subject has not previously received anantibiotic compound in the one month prior to administration of thetherapeutic composition. In other embodiments, the mammalian subject hasreceived one or more treatments with one or more different antibioticcompounds and such treatment(s) resulted in no improvement or aworsening of symptoms. In some embodiments, the spore composition isadministered following a successful course of antibiotics to preventdysbiosis and enhance recovery of a diverse, healthy microbiota.

In some embodiments, the gastrointestinal disease, disorder or conditionis diarrhea caused by C. difficile including recurrent C. difficileinfection, ulcerative colitis, colitis, Crohn's disease, or irritablebowel disease. Beneficially, the therapeutic composition is administeredonly once prior to improvement of the disease, disorder or condition. Insome embodiments the therapeutic composition is administered atintervals greater than two days, such as once every three, four, five orsix days, or every week or less frequently than every week. Or thepreparation may be administered intermittently according to a setschedule, e.g., once a day, once weekly, or once monthly, or when thesubject relapses from the primary illness. In another embodiment, thepreparation may be administered on a long-term basis to individuals whoare at risk for infection with or who may be carriers of thesepathogens, including individuals who will have an invasive medicalprocedure (such as surgery), who will be hospitalized, who live in along-term care or rehabilitation facility, who are exposed to pathogensby virtue of their profession (livestock and animal processing workers),or who could be carriers of pathogens (including hospital workers suchas physicians, nurses, and other healthcare professionals).

Also provided are methods of treating or preventing a mammalian subjectsuffering from or at risk of developing a metabolic disease, anddisorder or condition selected from the group consisting of diabetes,metabolic syndrome, obesity, heart disease, autoimmune disease, liverdisease, and autism using the therapeutic compositions provided herein.

In embodiments, the bacterial spore composition is administeredenterically. This preferentially includes oral administration, or by anoral or nasal tube (including nasogastric, nasojejunal, oral gastric, ororal jejunal). In other embodiments, administration includes rectaladministration (including enema, suppository, or colonoscopy). Thebacterial composition may be administered to at least one region of thegastrointestinal tract, including the mouth, esophagus, stomach, smallintestine, large intestine, and rectum. In some embodiments, it isadministered to all regions of the gastrointestinal tract. The bacterialcompositions may be administered orally in the form of medicaments suchas powders, capsules, tablets, gels or liquids. The bacterialcompositions may also be administered in gel or liquid form by the oralroute or through a nasogastric tube, or by the rectal route in a gel orliquid form, by enema or instillation through a colonoscope or by asuppository.

If the composition is administered colonoscopically and, optionally, ifthe bacterial composition is administered by other rectal routes (suchas an enema or suppository) or even if the subject has an oraladministration, the subject may have a colonic-cleansing preparation.The colon-cleansing preparation can facilitate proper use of thecolonoscope or other administration devices, but even when it does notserve a mechanical purpose it can also maximize the proportion of thebacterial composition relative to the other organisms previouslyresiding in the gastrointestinal tract of the subject. Any ordinarilyacceptable colonic-cleansing preparation may be used such as thosetypically provided when a subject undergoes a colonoscopy.

To evaluate the subject, symptoms of dysbiosis are evaluated posttreatment ranging from 1 day to 6 months after administration of thepurified spore population. Fecal material is collected during thisperiod and the microbes present in the gastrointestinal tract can beassessed by 16S rDNA or metagenomic sequencing analysis or otheranalyses commonly used by the skilled artisan. Repopulation by speciesprovided by the spore population as well as Augmentation by commensalmicrobes not present in the spore population will occur in this time asthe spore population catalyzes a reshaping of the gut ecology to a stateof healthy biosis. The specification is most thoroughly understood inlight of the teachings of the references cited within the specification.The embodiments within the specification provide an illustration ofembodiments and should not be construed to limit the scope. The skilledartisan readily recognizes that many other embodiments are encompassed.

All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts or is inconsistent with this specification, thespecification will supersede any such material. The citation of anyreferences herein is not an admission that such references are priorart.

Methods of Treating a Subject

In some embodiments, the compositions disclosed herein are administeredto a patient or a user (sometimes collectively referred to as a“subject”). As used herein “administer” and “administration” encompassesembodiments in which one person directs another to consume a bacterialcomposition in a certain manner and/or for a certain purpose, and alsosituations in which a user uses a bacteria composition in a certainmanner and/or for a certain purpose independently of or in variance toany instructions received from a second person. Non-limiting examples ofembodiments in which one person directs another to consume a bacterialcomposition in a certain manner and/or for a certain purpose includewhen a physician prescribes a course of conduct and/or treatment to apatient, when a parent commands a minor user (such as a child) toconsume a bacterial composition, when a trainer advises a user (such asan athlete) to follow a particular course of conduct and/or treatment,and when a manufacturer, distributer, or marketer recommends conditionsof use to an end user, for example through advertisements or labeling onpackaging or on other materials provided in association with the sale ormarketing of a product.

The bacterial compositions offer a protective and/or therapeutic effectagainst infection by one or more GI pathogens of interest and can beadministered after an acute case of infection has been resolved in orderto prevent relapse, during an acute case of infection as a complement toantibiotic therapy if the bacterial composition is not sensitive to thesame antibiotics as the GI pathogen, or to prevent infection or reducetransmission from disease carriers.

The present bacterial compositions can be useful in a variety ofclinical situations. For example, the bacterial compositions can beadministered as a complementary treatment to antibiotics when a patientis suffering from an acute infection, to reduce the risk of recurrenceafter an acute infection has subsided, or when a patient will be inclose proximity to others with or at risk of serious gastrointestinalinfections (physicians, nurses, hospital workers, family members ofthose who are ill or hospitalized).

The present bacterial compositions can be administered to animals,including humans, laboratory animals (e.g., primates, rats, mice),livestock (e.g., cows, sheep, goats, pigs, turkeys, chickens), andhousehold pets (e.g., dogs, cats, rodents).

In the present method, the bacterial composition can be administeredenterically, in other words, by a route of access to thegastrointestinal tract. This includes oral administration, rectaladministration (including enema, suppository, or colonoscopy), by anoral or nasal tube (nasogastric, nasojejunal, oral gastric, or oraljejunal), as detailed more fully herein.

Pretreatment Protocols

Prior to administration of the bacterial composition, the patient canoptionally have a pretreatment protocol to prepare the gastrointestinaltract to receive the bacterial composition. In certain embodiments, thepretreatment protocol is advisable, such as when a patient has an acuteinfection with a highly resilient pathogen. In other embodiments, thepretreatment protocol is entirely optional, such as when the pathogencausing the infection is not resilient, or the patient has had an acuteinfection that has been successfully treated but where the physician isconcerned that the infection may recur. In these instances, thepretreatment protocol can enhance the ability of the bacterialcomposition to affect the patient's microbiome.

As one way of preparing the patient for administration of the microbialecosystem, at least one antibiotic can be administered to alter thebacteria in the patient. As another way of preparing the patient foradministration of the microbial ecosystem, a standard colon-cleansingpreparation can be administered to the patient to substantially emptythe contents of the colon, such as used to prepare a patient for acolonscopy. By “substantially emptying the contents of the colon,” thisapplication means removing at least 75%, at least 80%, at least 90%, atleast 95%, or about 100% of the contents of the ordinary volume of coloncontents. Antibiotic treatment can precede the colon-cleansing protocol.

If a patient has received an antibiotic for treatment of an infection,or if a patient has received an antibiotic as part of a specificpretreatment protocol, in one embodiment, the antibiotic can be stoppedin sufficient time to allow the antibiotic to be substantially reducedin concentration in the gut before the bacterial composition isadministered. In one embodiment, the antibiotic can be discontinued 1,2, or 3 days before the administration of the bacterial composition. Inanother embodiment, the antibiotic can be discontinued 3, 4, 5, 6, or 7antibiotic half-lives before administration of the bacterialcomposition. In another embodiment, the antibiotic can be chosen so theconstituents in the bacterial composition have an MIC50 that is higherthan the concentration of the antibiotic in the gut.

MIC50 of a bacterial composition or the elements in the composition canbe determined by methods well known in the art. Reller et al.,Antimicrobial Susceptibility Testing: A Review of General Principles andContemporary Practices, Clinical Infectious Diseases 49(11):1749-1755(2009). In such an embodiment, the additional time between antibioticadministration and administration of the bacterial composition is notnecessary. If the pretreatment protocol is part of treatment of an acuteinfection, the antibiotic can be chosen so that the infection issensitive to the antibiotic, but the constituents in the bacterialcomposition are not sensitive to the antibiotic.

Routes of Administration

The bacterial compositions of the invention are suitable foradministration to mammals and non-mammalian animals in need thereof. Incertain embodiments, the mammalian subject is a human subject who hasone or more symptoms of a dysbiosis.

When a mammalian subject is suffering from a disease, disorder orcondition characterized by an aberrant microbiota, the bacterialcompositions described herein are suitable for treatment thereof. Insome embodiments, the mammalian subject has not received antibiotics inadvance of treatment with the bacterial compositions. For example, themammalian subject has not been administered at least two doses ofvancomycin, metronidazole and/or or similar antibiotic compound withinone week prior to administration of the therapeutic composition. Inother embodiments, the mammalian subject has not previously received anantibiotic compound in the one month prior to administration of thetherapeutic composition. In other embodiments, the mammalian subject hasreceived one or more treatments with one or more different antibioticcompounds and such treatment(s) resulted in no improvement or aworsening of symptoms.

In some embodiments, the gastrointestinal disease, disorder or conditionis diarrhea caused by C. difficile including recurrent C. difficileinfection, ulcerative colitis, colitis, Crohn's disease, or irritablebowel disease. Beneficially, the therapeutic composition is administeredonly once prior to improvement of the disease, disorder or condition. Insome embodiments, the therapeutic composition is administered atintervals greater than two days, such as once every three, four, five orsix days, or every week or less frequently than every week. In otherembodiments, the preparation can be administered intermittentlyaccording to a set schedule, e.g., once a day, once weekly, or oncemonthly, or when the subject relapses from the primary illness. Inanother embodiment, the preparation may be administered on a long-termbasis to subjects who are at risk for infection with or who may becarriers of these pathogens, including subjects who will have aninvasive medical procedure (such as surgery), who will be hospitalized,who live in a long-term care or rehabilitation facility, who are exposedto pathogens by virtue of their profession (livestock and animalprocessing workers), or who could be carriers of pathogens (includinghospital workers such as physicians, nurses, and other health careprofessionals).

In certain embodiments, the bacterial composition is administeredenterically. This preferentially includes oral administration, or by anoral or nasal tube (including nasogastric, nasojejunal, oral gastric, ororal jejunal). In other embodiments, administration includes rectaladministration (including enema, suppository, or colonoscopy). Thebacterial composition can be administered to at least one region of thegastrointestinal tract, including the mouth, esophagus, stomach, smallintestine, large intestine, and rectum. In some embodiments, it isadministered to all regions of the gastrointestinal tract. The bacterialcompositions can be administered orally in the form of medicaments suchas powders, capsules, tablets, gels or liquids. The bacterialcompositions can also be administered in gel or liquid form by the oralroute or through a nasogastric tube, or by the rectal route in a gel orliquid form, by enema or instillation through a colonoscope or by asuppository.

If the composition is administered colonoscopically and, optionally, ifthe bacterial composition is administered by other rectal routes (suchas an enema or suppository) or even if the subject has an oraladministration, the subject can have a colon-cleansing preparation. Thecolon-cleansing preparation can facilitate proper use of the colonoscopeor other administration devices, but even when it does not serve amechanical purpose, it can also maximize the proportion of the bacterialcomposition relative to the other organisms previously residing in thegastrointestinal tract of the subject. Any ordinarily acceptablecolon-cleansing preparation may be used such as those typically providedwhen a subject undergoes a colonoscopy.

Dosages and Schedule for Administration

In some embodiments, the bacteria and bacterial compositions areprovided in a dosage form. In certain embodiments, the dosage form isdesigned for administration of at least one OTU or combination thereofdisclosed herein, wherein the total amount of bacterial compositionadministered is selected from 0.1 ng to 10 g, 10 ng to 1 g, 100 ng to0.1 g, 0.1 mg to 500 mg, 1 mg to 100 mg, or from 10-15 mg. In otherembodiments, the bacterial composition is consumed at a rate of from 0.1ng to 10 g a day, 10 ng to 1 g a day, 100 ng to 0.1 g a day, 0.1 mg to500 mg a day, 1 mg to 100 mg a day, or from 10-15 mg a day, or more.

In certain embodiments, the treatment period is at least 1 day, at least2 days, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4weeks, at least 1 month, at least 2 months, at least 3 months, at least4 months, at least 5 months, at least 6 months, or at least 1 year. Insome embodiments the treatment period is from 1 day to 1 week, from 1week to 4 weeks, from 1 month, to 3 months, from 3 months to 6 months,from 6 months to 1 year, or for over a year.

In one embodiment, 105 and 1012 microorganisms total can be administeredto the patient in a given dosage form. In another embodiment, aneffective amount can be provided in from 1 to 500 ml or from 1 to 500grams of the bacterial composition having from 107 to 1011 bacteria perml or per gram, or a capsule, tablet or suppository having from 1 mg to1000 mg lyophilized powder having from 107 to 1011 bacteria. Thosereceiving acute treatment can receive higher doses than those who arereceiving chronic administration (such as hospital workers or thoseadmitted into long-term care facilities).

Any of the preparations described herein can be administered once on asingle occasion or on multiple occasions, such as once a day for severaldays or more than once a day on the day of administration (includingtwice daily, three times daily, or up to five times daily). In anotherembodiment, the preparation can be administered intermittently accordingto a set schedule, e.g., once weekly, once monthly, or when the patientrelapses from the primary illness. In one embodiment, the preparationcan be administered on a long-term basis to individuals who are at riskfor infection with or who may be carriers of these pathogens, includingindividuals who will have an invasive medical procedure (such assurgery), who will be hospitalized, who live in a long-term care orrehabilitation facility, who are exposed to pathogens by virtue of theirprofession (livestock and animal processing workers), or who could becarriers of pathogens (including hospital workers such as physicians,nurses, and other health care professionals).

Patient Selection

Particular bacterial compositions can be selected for individualpatients or for patients with particular profiles. For example, 16Ssequencing can be performed for a given patient to identify the bacteriapresent in his or her microbiota. The sequencing can either profile thepatient's entire microbiome using 16S sequencing (to the family, genera,or species level), a portion of the patient's microbiome using 16Ssequencing, or it can be used to detect the presence or absence ofspecific candidate bacteria that are biomarkers for health or aparticular disease state, such as markers of multi-drug resistantorganisms or specific genera of concern such as Escherichia. Based onthe biomarker data, a particular composition can be selected foradministration to a patient to supplement or complement a patient'smicrobiota in order to restore health or treat or prevent disease. Inanother embodiment, patients can be screened to determine thecomposition of their microbiota to determine the likelihood ofsuccessful treatment.

Combination Therapy

The bacterial compositions can be administered with other agents in acombination therapy mode, including anti-microbial agents andprebiotics. Administration can be sequential, over a period of hours ordays, or simultaneous.

In one embodiment, the bacterial compositions are included incombination therapy with one or more anti-microbial agents, whichinclude anti-bacterial agents, anti-fungal agents, anti-viral agents andanti-parasitic agents.

Anti-bacterial agents can include cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

Anti-viral agents can include Abacavir, Acyclovir, Adefovir, Amprenavir,Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol,Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine,Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine,Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Nevirapine,Penciclovir, Raltegravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine,Tenofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine,Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir,Zalcitabine, Zanamivir and Zidovudine.

Examples of antifungal compounds include, but are not limited to polyeneantifungals such as natamycin, rimocidin, filipin, nystatin,amphotericin B, candicin, and hamycin; imidazole antifungals such asmiconazole, ketoconazole, clotrimazole, econazole, omoconazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, and tioconazole; triazole antifungals suchas fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole,voriconazole, terconazole, and albaconazole; thiazole antifungals suchas abafungin; allylamine antifungals such as terbinafine, naftifine, andbutenafine; and echinocandin antifungals such as anidulafungin,caspofungin, and micafungin. Other compounds that have antifungalproperties include, but are not limited to polygodial, benzoic acid,ciclopirox, tolnaftate, undecylenic acid, flucytosine or5-fluorocytosine, griseofulvin, and haloprogin.

In one embodiment, the bacterial compositions are included incombination therapy with one or more corticosteroids, mesalazine,mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressivedrugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone,methotrexate, antihistamines, glucocorticoids, epinephrine,theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugsfor rhinitis, anti-cholinergic decongestants, mast-cell stabilizers,monoclonal anti-IgE antibodies, vaccines, and combinations thereof.

A prebiotic is a selectively fermented ingredient that allows specificchanges, both in the composition and/or activity in the gastrointestinalmicrobiota that confers benefits upon host well-being and health.Prebiotics can include complex carbohydrates, amino acids, peptides, orother essential nutritional components for the survival of the bacterialcomposition. Prebiotics include, but are not limited to, amino acids,biotin, fructooligosaccharide, galactooligosaccharides, inulin,lactulose, mannan oligosaccharides, oligofructose-enriched inulin,oligofructose, oligodextrose, tagatose, trans-galactooligosaccharide,and xylooligosaccharides.

Methods for Testing Bacterial Compositions for Populating Effect

In Vivo Assay for Determining Whether a Bacterial Composition Populatesa Subject's Gastrointestinal Tract

In order to determine that the bacterial composition populates thegastrointestinal tract of a subject, an animal model, such as a mousemodel, can be used. The model can begin by evaluating the microbiota ofthe mice. Qualitative assessments can be accomplished using 16Sprofiling of the microbial community in the feces of normal mice. It canalso be accomplished by full genome sequencing, whole genome shotgunsequencing (WGS), or traditional microbiological techniques.Quantitative assessments can be conducted using quantitative PCR (qPCR),described below, or by using traditional microbiological techniques andcounting colony formation.

Optionally, the mice can receive an antibiotic treatment to mimic thecondition of dysbiosis. Antibiotic treatment can decrease the taxonomicrichness, diversity, and evenness of the community, including areduction of abundance of a significant number of bacterial taxa.Dethlefsen et al., The pervasive effects of an antibiotic on the humangut microbiota, as revealed by deep 16S rRNA sequencing, PLoS Biology6(11):3280 (2008). At least one antibiotic can be used, and antibioticsare well known. Antibiotics can include aminoglycoside antibiotic(amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, rhodostreptomycin, streptomycin, tobramycin, andapramycin), amoxicillin, ampicillin, Augmentin (anamoxicillin/clavulanate potassium combination), cephalosporin (cefaclor,defadroxil, cefazolin, cefixime, fefoxitin, cefprozil, ceftazimdime,cefuroxime, cephalexin), clavulanate potassium, clindamycin, colistin,gentamycin, kanamycin, metronidazole, or vancomycin. As an individual,nonlimiting specific example, the mice can be provided with drinkingwater containing a mixture of the antibiotics kanamycin, colistin,gentamycin, metronidazole and vancomycin at 40 mg/kg, 4.2 mg/kg, 3.5mg/kg, 21.5 mg/kg, and 4.5 mg/kg (mg per average mouse body weight),respectively, for 7 days. Alternatively, mice can be administeredciprofloxacin at a dose of 15-20 mg/kg (mg per average mouse bodyweight), for 7 days. If the mice are provided with an antibiotic, a washout period of from one day to three days may be provided with noantibiotic treatment and no bacterial composition treatment.

Subsequently, the test bacterial composition is administered to the miceby oral gavage. The test bacterial composition may be administered in avolume of 0.2 ml containing 10⁴ CFUs of each type of bacteria in thebacterial composition. Dose-response may be assessed by using a range ofdoses, including, but not limited to 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, and/or 10¹⁰.

The mice can be evaluated using 16S sequencing, full genome sequencing,whole genome shotgun sequencing (WGS), or traditional microbiologicaltechniques to determine whether the test bacterial composition haspopulated the gastrointestinal tract of the mice. For example only, oneday, three days, one week, two weeks, and one month after administrationof the bacterial composition to the mice, 16S profiling is conducted todetermine whether the test bacterial composition has populated thegastrointestinal tract of the mice. Quantitative assessments, includingqPCR and traditional microbiological techniques such as colony counting,can additionally or alternatively be performed, at the same timeintervals.

Furthermore, the number of sequence counts that correspond exactly tothose in the bacterial composition over time can be assessed todetermine specifically which components of the bacterial compositionreside in the gastrointestinal tract over a particular period of time.In one embodiment, the strains of the bacterial composition persist fora desired period of time. In another embodiment, the components of thebacterial composition persist for a desired period of time, while alsoincreasing the ability of other microbes (such as those present in theenvironment, food, etc.) to populate the gastrointestinal tract, furtherincreasing overall diversity, as discussed below.

Ability of Bacterial Compositions to Populate Different Regions of theGastrointestinal Tract

The present bacterial compositions can also be assessed for theirability to populate different regions on the gastrointestinal tract. Inone embodiment, a bacterial composition can be chosen for its ability topopulate one or more than one region of the gastrointestinal tract,including, but not limited to the stomach, the small intestine(duodenum, jejunum, and ileum), the large intestine (the cecum, thecolon (the ascending, transverse, descending, and sigmoid colon), andthe rectum).

An in vivo study can be conducted to determine which regions of thegastrointestinal tract a given bacterial composition will populate. Amouse model similar to the one described above can be conducted, exceptinstead of assessing the feces produced by the mice, particular regionsof the gastrointestinal tract can be removed and studied individually.For example, at least one particular region of the gastrointestinaltract can be removed and a qualitative or quantitative determination canbe performed on the contents of that region of the gastrointestinaltract. In another embodiment, the contents can optionally be removed andthe qualitative or quantitative determination may be conducted on thetissue removed from the mouse.

qPCR

As one quantitative method for determining whether a bacterialcomposition populates the gastrointestinal tract, quantitative PCR(qPCR) can be performed. Standard techniques can be followed to generatea standard curve for the bacterial composition of interest, either forall of the components of the bacterial composition collectively,individually, or in subsets (if applicable). Genomic DNA can beextracted from samples using commercially-available kits, such as the MoBio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

In some embodiments, qPCR can be conducted using HotMasterMix (5PRIME,Gaithersburg, Md.) and primers specific for the bacterial composition ofinterest, and may be conducted on a MicroAmp Fast Optical 96-wellReaction Plate with Barcode (0.1 mL) (Life Technologies, Grand Island,N.Y.) and performed on a BioRad C1000™ Thermal Cycler equipped with aCFX96™ Real-Time System (BioRad, Hercules, Calif.), with fluorescentreadings of the FAM and ROX channels. The Cq value for each well on theFAM channel is determined by the CFX Manager™ software version 2.1. Thelog₁₀ (cfu/ml) of each experimental sample is calculated by inputting agiven sample's Cq value into linear regression model generated from thestandard curve comparing the Cq values of the standard curve wells tothe known log₁₀ (cfu/ml) of those samples. The skilled artisan mayemploy alternative qPCR modes.

Methods for Characterization of Bacterial Compositions

In certain embodiments, provided are methods for testing certaincharacteristics of bacterial compositions. For example, the sensitivityof bacterial compositions to certain environmental variables isdetermined, e.g., in order to select for particular desirablecharacteristics in a given composition, formulation and/or use. Forexample, the constituents in the bacterial composition can be tested forpH resistance, bile acid resistance, and/or antibiotic sensitivity,either individually on a constituent-by-constituent basis orcollectively as a bacterial composition comprised of multiple bacterialconstituents (collectively referred to in this section as bacterialcomposition).

pH Sensitivity Testing

If a bacterial composition will be administered other than to the colonor rectum (i.e., for example, an oral route), optionally testing for pHresistance enhances the selection of bacterial compositions that willsurvive at the highest yield possible through the varying pHenvironments of the distinct regions of the GI tract. Understanding howthe bacterial compositions react to the pH of the GI tract also assistsin formulation, so that the number of bacteria in a dosage form can beincreased if beneficial and/or so that the composition may beadministered in an enteric-coated capsule or tablet or with a bufferingor protective composition. As the pH of the stomach can drop to a pH of1 to 2 after a high-protein meal for a short time before physiologicalmechanisms adjust it to a pH of 3 to 4 and often resides at a resting pHof 4 to 5, and as the pH of the small intestine can range from a pH of 6to 7.4, bacterial compositions can be prepared that survive thesevarying pH ranges (specifically wherein at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or as much as 100% of thebacteria can survive gut transit times through various pH ranges). Thiscan be tested by exposing the bacterial composition to varying pH rangesfor the expected gut transit times through those pH ranges. Therefore,as a nonlimiting example only, 18-hour cultures of bacterialcompositions can be grown in standard media, such as gut microbiotamedium (“GMM”, see Goodman et al., Extensive personal human gutmicrobiota culture collections characterized and manipulated ingnotobiotic mice, PNAS 108(15):6252-6257 (2011)) or anotheranimal-products-free medium, with the addition of pH adjusting agentsfor a pH of 1 to 2 for 30 minutes, a pH of 3 to 4 for 1 hour, a pH of 4to 5 for 1 to 2 hours, and a pH of 6 to 7.4 for 2.5 to 3 hours. Analternative method for testing stability to acid is described in U.S.Pat. No. 4,839,281. Survival of bacteria may be determined by culturingthe bacteria and counting colonies on appropriate selective ornon-selective media.

Bile Acid Sensitivity Testing

Additionally, in some embodiments, testing for bile-acid resistanceenhances the selection of bacterial compositions that will surviveexposures to bile acid during transit through the GI tract. Bile acidsare secreted into the small intestine and can, like pH, affect thesurvival of bacterial compositions. This can be tested by exposing thebacterial compositions to bile acids for the expected gut exposure timeto bile acids. For example, bile acid solutions can be prepared atdesired concentrations using 0.05 mM Tris at pH 9 as the solvent. Afterthe bile acid is dissolved, the pH of the solution may be adjusted to7.2 with 10% HCl. Bacterial compositions can be cultured in 2.2 ml of abile acid composition mimicking the concentration and type of bile acidsin the patient, 1.0 ml of 10% sterile-filtered feces media and 0.1 ml ofan 18-hour culture of the given strain of bacteria. Incubations may beconducted for from 2.5 to 3 hours or longer. An alternative method fortesting stability to bile acid is described in U.S. Pat. No. 4,839,281.Survival of bacteria may be determined by culturing the bacteria andcounting colonies on appropriate selective or non-selective media.

Antibiotic Sensitivity Testing

As a further optional sensitivity test, bacterial compositions can betested for sensitivity to antibiotics. In one embodiment, bacterialcompositions can be chosen so that the bacterial constituents aresensitive to antibiotics such that if necessary they can be eliminatedor substantially reduced from the patient's gastrointestinal tract by atleast one antibiotic targeting the bacterial composition.

Adherence to Gastrointestinal Cells

The bacterial compositions may optionally be tested for the ability toadhere to gastrointestinal cells. A method for testing adherence togastrointestinal cells is described in U.S. Pat. No. 4,839,281.

Methods for Purifying Spores

Solvent Treatments

To purify the bacterial spores, the fecal material is subjected to oneor more solvent treatments. A solvent treatment is a miscible solventtreatment (either partially miscible or fully miscible) or an immisciblesolvent treatment. Miscibility is the ability of two liquids to mix witheach to form a homogeneous solution. Water and ethanol, for example, arefully miscible such that a mixture containing water and ethanol in anyratio will show only one phase. Miscibility is provided as a wt/wt %, orweight of one solvent in 100 g of final solution. If two solvents arefully miscible in all proportions, their miscibility is 100%. Providedas fully miscible solutions with water are alcohols, e.g., methanol,ethanol, isopropanol, butanol, etc. The alcohols can be provided alreadycombined with water; e.g., a solution containing 10%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 89%, 85%, 90%, 95% orgreater than 95% Other solvents are only partially miscible, meaningthat only some portion will dissolve in water. Diethyl ether, forexample, is partially miscible with water. Up to 7 grams of diethylether will dissolve in 93 g of water to give a 7% (wt/wt %) solution. Ifmore diethyl ether is added, a two phase solution will result with adistinct diethyl ether layer above the water. Other miscible materialsinclude ethers, dimethoxyethane, or tetrahydrofuran In contrast, an oilsuch as an alkane and water are immiscible and form two phases. Further,immiscible treatments are optionally combined with a detergent, eitheran ionic detergent or a non-ionic detergent. Exemplary detergentsinclude Triton X-100, Tween 20, Tween 80, Nonidet P40, a pluronic, orapolyol.

Chromatography Treatments

To purify spore populations, the fecal materials are subjected to one ormore chromatographic treatments, either sequentially or in parallel. Ina chromatographic treatment, a solution containing the fecal material iscontacted with a solid medium containing a hydrophobic interactionchromatographic (HIC) medium or an affinity chromatographic medium. Inan alternative embodiment, a solid medium capable of absorbing aresidual habitat product present in the fecal material is contacted witha solid medium that adsorbs a residual habitat product. In certainembodiments, the HIC medium contains sepharose or a derivatizedsepharose such as butyl sepharose, octyl sepharose, phenyl sepharose, orbutyl-s sepharose. In other embodiments, the affinity chromatographicmedium contains material derivatized with mucin type I, II, III, IV, V,or VI, or oligosaccharides derived from or similar to those of mucinstype I, II, III, IV, V, or VI. Alternatively, the affinitychromatographic medium contains material derivatized with antibodiesthat recognize spore-forming bacteria.

Mechanical Treatments

Provided herein is the physical disruption of the fecal material,particularly by one or more mechanical treatment such as blending,mixing, shaking, vortexing, impact pulverization, and sonication. Asprovided herein, the mechanical disrupting treatment substantiallydisrupts a non-spore material present in the fecal material and does notsubstantially disrupt a spore present in the fecal material. Mechanicaltreatments optionally include filtration treatments, where the desiredspore populations are retained on a filter while the undesirable(non-spore) fecal components to pass through, and the spore fraction isthen recovered from the filter medium. Alternatively, undesirableparticulates and eukaryotic cells may be retained on a filter whilebacterial cells including spores pass through. In some embodiments thespore fraction retained on the filter medium is subjected to adiafiltration step, wherein the retained spores are contacted with awash liquid, typically a sterile saline-containing solution or otherdiluent, in order to further reduce or remove the undesirable fecalcomponents.

Thermal Treatments

Provided herein is the thermal disruption of the fecal material.Generally, the fecal material is mixed in a saline-containing solutionsuch as phosphate-buffered saline (PBS) and subjected to a heatedenvironment, such as a warm room, incubator, water-bath, or the like,such that efficient heat transfer occurs between the heated environmentand the fecal material. Preferably the fecal material solution is mixedduring the incubation to enhance thermal conductivity and disruptparticulate aggregates. Thermal treatments can be modulated by thetemperature of the environment and/or the duration of the thermaltreatment. For example, the fecal material or a liquid comprising thefecal material is subjected to a heated environment, e.g., a hot waterbath of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 or greater than 100 degrees Celsius, for at leastabout 1, 5, 10, 15, 20, 30, 45 seconds, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more than 10 hours. In certain embodiments the thermal treatmentoccurs at two different temperatures, such as 30 seconds in a 100 degreeCelsius environment followed by 10 minutes in a 50 degree Celsiusenvironment. In preferred embodiments the temperature and duration ofthe thermal treatment are sufficient to kill or remove pathogenicmaterials while not substantially damaging or reducing thegermination-competency of the spores.

Irradiation Treatments

Provided are methods of treating the fecal material or separatedcontents of the fecal material with ionizing radiation, typically gammairradiation, ultraviolet irradiation or electron beam irradiationprovided at an energy level sufficient to kill pathogenic materialswhile not substantially damaging the desired spore populations. Forexample, ultraviolet radiation at 254 nm provided at an energy levelbelow about 22,000 microwatt seconds per cm² will not generally destroydesired spores.

Centrifugation and Density Separation Treatments

Provided are methods of separating desired spore populations from theother components of the fecal material by centrifugation. A solutioncontaining the fecal material is subjected to one or more centrifugationtreatments, e.g., at about 1000×g, 2000×g, 3000×g, 4000×g, 5000×g,6000×g, 7000×g, 8000×g or greater than 8000×g. Differentialcentrifugation separates desired spores from undesired non-sporematerial; at low forces the spores are retained in solution, while athigher forces the spores are pelleted while smaller impurities (e.g.,virus particles, phage) are retained in solution. For example, a firstlow force centrifugation pellets fibrous materials; a second, higherforce centrifugation pellets undesired eukaryotic cells, and a third,still higher force centrifugation pellets the desired spores while smallcontaminants remain in suspension. In some embodiments density ormobility gradients or cushions (e.g., step cushions), such as Percoll,Ficoll, Nycodenz, Histodenz or sucrose gradients, are used to separatedesired spore populations from other materials in the fecal material.

Also provided herein are methods of producing spore populations thatcombine two or more of the treatments described herein in order tosynergistically purify the desired spores while killing or removingundesired materials and/or activities from the spore population. It isgenerally desirable to retain the spore populations undernon-germinating and non-growth promoting conditions and media, in orderto minimize the growth of pathogenic bacteria present in the sporepopulations and to minimize the germination of spores into vegetativebacterial cells.

Pharmaceutical Compositions and Formulations of the Invention

Formulations

Provided are formulations for administration to humans and othersubjects in need thereof. Generally the bacterial compositions arecombined with additional active and/or inactive materials in order toproduce a final product, which may be in single dosage unit or in amulti-dose format.

In some embodiments, the composition comprises at least onecarbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars.The terms “saccharide,” “polysaccharide,” “carbohydrate,” and“oligosaccharide” may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate can be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates can contain modified saccharideunits, such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates can exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. Asused herein, a “lipid” includes fats, oils, triglycerides, cholesterol,phospholipids, fatty acids in any form including free fatty acids. Fats,oils and fatty acids can be saturated, unsaturated (cis or trans) orpartially unsaturated (cis or trans). In some embodiments, the lipidcomprises at least one fatty acid selected from lauric acid (12:0),myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1),margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0),oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3),octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid(20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4),eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoicacid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6)(DHA), and tetracosanoic acid (24:0). In other embodiments, thecomposition comprises at least one modified lipid, for example, a lipidthat has been modified by cooking.

In some embodiments, the composition comprises at least one supplementalmineral or mineral source. Examples of minerals include, withoutlimitation: chloride, sodium, calcium, iron, chromium, copper, iodine,zinc, magnesium, manganese, molybdenum, phosphorus, potassium, andselenium. Suitable forms of any of the foregoing minerals includesoluble mineral salts, slightly soluble mineral salts, insoluble mineralsalts, chelated minerals, mineral complexes, non-reactive minerals suchas carbonyl minerals, and reduced minerals, and combinations thereof.

In certain embodiments, the composition comprises at least onesupplemental vitamin. The at least one vitamin can be fat-soluble orwater soluble vitamins. Suitable vitamins include but are not limited tovitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin,niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine,pantothenic acid, and biotin. Suitable forms of any of the foregoing aresalts of the vitamin, derivatives of the vitamin, compounds having thesame or similar activity of the vitamin, and metabolites of the vitamin.

In other embodiments, the composition comprises an excipient.Non-limiting examples of suitable excipients include a buffering agent,a preservative, a stabilizer, a binder, a compaction agent, a lubricant,a dispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In another embodiment, the excipient is a buffering agent. Non-limitingexamples of suitable buffering agents include sodium citrate, magnesiumcarbonate, magnesium bicarbonate, calcium carbonate, and calciumbicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In other embodiments, the composition comprises a binder as anexcipient. Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C₁₂-C₁₈ fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In another embodiment, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In other embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin,bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In other embodiments, the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, micro-crystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pecitin, andtragacanth. In another embodiment, the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In another embodiment, the excipient comprises a flavoring agent.Flavoring agents can be chosen from synthetic flavor oils and flavoringaromatics; natural oils; extracts from plants, leaves, flowers, andfruits; and combinations thereof. In some embodiments the flavoringagent is selected from cinnamon oils; oil of wintergreen; peppermintoils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oilsuch as lemon oil, orange oil, grape and grapefruit oil; and fruitessences including apple, peach, pear, strawberry, raspberry, cherry,plum, pineapple, and apricot.

In other embodiments, the excipient comprises a sweetener. Non-limitingexamples of suitable sweeteners include glucose (corn syrup), dextrose,invert sugar, fructose, and mixtures thereof (when not used as acarrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; and sugar alcohols such as sorbitol,mannitol, sylitol, and the like. Also contemplated are hydrogenatedstarch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In yet other embodiments, the composition comprises a coloring agent.Non-limiting examples of suitable color agents include food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), and externaldrug and cosmetic colors (Ext. D&C). The coloring agents can be used asdyes or their corresponding lakes.

The weight fraction of the excipient or combination of excipients in theformulation is usually about 99% or less, such as about 95% or less,about 90% or less, about 85% or less, about 80% or less, about 75% orless, about 70% or less, about 65% or less, about 60% or less, about 55%or less, 50% or less, about 45% or less, about 40% or less, about 35% orless, about 30% or less, about 25% or less, about 20% or less, about 15%or less, about 10% or less, about 5% or less, about 2% or less, or about1% or less of the total weight of the composition.

The bacterial compositions disclosed herein can be formulated into avariety of forms and administered by a number of different means. Thecompositions can be administered orally, rectally, or parenterally, informulations containing conventionally acceptable carriers, adjuvants,and vehicles as desired. The term “parenteral” as used herein includessubcutaneous, intravenous, intramuscular, or intrasternal injection andinfusion techniques. In an exemplary embodiment, the bacterialcomposition is administered orally.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, troches, lozenges, powders, and granules. A capsuletypically comprises a core material comprising a bacterial compositionand a shell wall that encapsulates the core material. In someembodiments, the core material comprises at least one of a solid, aliquid, and an emulsion. In other embodiments, the shell wall materialcomprises at least one of a soft gelatin, a hard gelatin, and a polymer.Suitable polymers include, but are not limited to: cellulosic polymerssuch as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose (HPMC), methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulosesuccinate and carboxymethylcellulose sodium; acrylic acid polymers andcopolymers, such as those formed from acrylic acid, methacrylic acid,methyl acrylate, ammonio methylacrylate, ethyl acrylate, methylmethacrylate and/or ethyl methacrylate (e.g., those copolymers soldunder the trade name “Eudragit”); vinyl polymers and copolymers such aspolyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate,vinylacetate crotonic acid copolymer, and ethylene-vinyl acetatecopolymers; and shellac (purified lac). In yet other embodiments, atleast one polymer functions as taste-masking agents.

Tablets, pills, and the like can be compressed, multiply compressed,multiply layered, and/or coated. The coating can be single or multiple.In one embodiment, the coating material comprises at least one of asaccharide, a polysaccharide, and glycoproteins extracted from at leastone of a plant, a fungus, and a microbe. Non-limiting examples includecorn starch, wheat starch, potato starch, tapioca starch, cellulose,hemicellulose, dextrans, maltodextrin, cyclodextrins, inulins, pectin,mannans, gum arabic, locust bean gum, mesquite gum, guar gum, gumkaraya, gum ghatti, tragacanth gum, funori, carrageenans, agar,alginates, chitosans, or gellan gum. In some embodiments the coatingmaterial comprises a protein. In another embodiment, the coatingmaterial comprises at least one of a fat and an oil. In otherembodiments, the at least one of a fat and an oil is high temperaturemelting. In yet another embodiment, the at least one of a fat and an oilis hydrogenated or partially hydrogenated. In one embodiment, the atleast one of a fat and an oil is derived from a plant. In otherembodiments, the at least one of a fat and an oil comprises at least oneof glycerides, free fatty acids, and fatty acid esters. In someembodiments, the coating material comprises at least one edible wax. Theedible wax can be derived from animals, insects, or plants. Non-limitingexamples include beeswax, lanolin, bayberry wax, carnauba wax, and ricebran wax. Tablets and pills can additionally be prepared with entericcoatings.

Alternatively, powders or granules embodying the bacterial compositionsdisclosed herein can be incorporated into a food product. In someembodiments, the food product is a drink for oral administration.Non-limiting examples of a suitable drink include fruit juice, a fruitdrink, an artificially flavored drink, an artificially sweetened drink,a carbonated beverage, a sports drink, a liquid diary product, a shake,an alcoholic beverage, a caffeinated beverage, infant formula and soforth. Other suitable means for oral administration include aqueous andnonaqueous solutions, emulsions, suspensions and solutions and/orsuspensions reconstituted from non-effervescent granules, containing atleast one of suitable solvents, preservatives, emulsifying agents,suspending agents, diluents, sweeteners, coloring agents, and flavoringagents.

In some embodiments, the food product can be a solid foodstuff. Suitableexamples of a solid foodstuff include without limitation a food bar, asnack bar, a cookie, a brownie, a muffin, a cracker, an ice cream bar, afrozen yogurt bar, and the like.

In other embodiments, the compositions disclosed herein are incorporatedinto a therapeutic food. In some embodiments, the therapeutic food is aready-to-use food that optionally contains some or all essentialmacronutrients and micronutrients. In another embodiment, thecompositions disclosed herein are incorporated into a supplementary foodthat is designed to be blended into an existing meal. In one embodiment,the supplemental food contains some or all essential macronutrients andmicronutrients. In another embodiment, the bacterial compositionsdisclosed herein are blended with or added to an existing food tofortify the food's protein nutrition. Examples include food staples(grain, salt, sugar, cooking oil, margarine), beverages (coffee, tea,soda, beer, liquor, sports drinks), snacks, sweets and other foods.

In one embodiment, the formulations are filled into gelatin capsules fororal administration. An example of an appropriate capsule is a 250 mggelatin capsule containing from 10 (up to 100 mg) of lyophilized powder(10⁸ to 10¹¹ bacteria), 160 mg microcrystalline cellulose, 77.5 mggelatin, and 2.5 mg magnesium stearate. In an alternative embodiment,from 10⁵ to 10¹² bacteria may be used, 10⁵ to 10⁷, 10⁶ to 10⁷, or 10⁸ to10¹⁰, with attendant adjustments of the excipients if necessary. In analternative embodiment, an enteric-coated capsule or tablet or with abuffering or protective composition can be used.

Examples

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1. Provision of Fecal Material

Fresh fecal samples were obtained from healthy human donors who havebeen screened for general good health and for the absence of infectiousdiseases, and meet inclusion and exclusion criteria, inclusion criteriainclude being in good general health, without significant medicalhistory, physical examination findings, or clinical laboratoryabnormalities, regular bowel movements with stool appearance typicallyType 2, 3, 4, 5 or 6 on the Bristol Stool Scale, and having a BMI ≦18kg/m² and ≧25 kg/m². Exclusion criteria generally included significantchronic or acute medical conditions including renal, hepatic, pulmonary,gastrointestinal, cardiovascular, genitourinary, endocrine, immunologic,metabolic, neurologic or hematological disease, a family history of,inflammatory bowel disease including Crohn's disease and ulcerativecolitis, Irritable bowel syndrome, colon, stomach or othergastrointestinal malignancies, or gastrointestinal polyposis syndromes,or recent use of yogurt or commercial probiotic materials in which anorganism(s) is a primary component. Samples were collected directlyusing a commode specimen collection system, which contains a plasticsupport placed on the toilet seat and a collection container that restson the support. Feces were deposited into the container, and the lid wasthen placed on the container and sealed tightly. The sample was thendelivered on ice within 1-4 hours for processing. Samples were mixedwith a sterile disposable tool, and 2-4 g aliquots were weighed andplaced into tubes and flash frozen in a dry ice/ethanol bath. Aliquotsare frozen at −80 degrees Celsius until use.

Optionally, the fecal material was suspended in a solution, and/orfibrous and/or particulate materials were removed. A frozen aliquotcontaining a known weight of feces was removed from storage at −80degrees Celsius and allowed to thaw at room temperature. Sterile 1×PBSwas added to create a 10% w/v suspension, and vigorous vortexing wasperformed to suspend the fecal material until the material appearedhomogeneous. The material was then left to sit for 10 minutes at roomtemperature to sediment fibrous and particulate matter. The suspensionabove the sediment was then carefully removed into a new tube andcontains a purified spore population. Optionally, the suspension wasthen centrifuged at a low speed, e.g., 1000×g, for 5 minutes to pelletparticulate matter including fibers. The pellet was discarded and thesupernatant, which contained vegetative organisms and spores, wasremoved into a new tube. The supernatant was then centrifuged at 6000×gfor 10 minutes to pellet the vegetative organisms and spores. The pelletwas then resuspended in 1×PBS with vigorous vortexing until the materialappears homogenous.

Example 2. Spore Purification from Alcohol Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS was mixed withabsolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. Thesuspension was incubated at 37 degrees Celsius for 1 hour. Afterincubation the suspension was centrifuged at 13,000 rpm for 5 minutes topellet spores. The supernatant was discarded and the pellet wasresuspended in an equal volume of PBS. Glycerol was added to a finalconcentration of 15% and then the purified spore fraction is stored at−80 degrees Celsius.

Example 2A. Generation of a Spore Preparation from Alcohol Treatment ofFecal Material

A 10% w/v suspension of human fecal material in PBS was mixed withabsolute ethanol in a 1:1 ratio and vortexed to mix for 1 minute. Thesuspension was incubated at 37 degrees Celsius for 1 hour. Afterincubation the suspension is centrifuged at 13,000 rpm for 5 minutes toconcentrate spores into a pellet containing a purified sprore-containingpreparation. The supernatant was discarded and the pellet resuspended inan equal volume of PBS. Glycerol was added to a final concentration of15% and then the purified spore preparation was stored at −80 degreesCelsius.

Example 3. Spore Purification from Thermal Treatment of Fecal Material

A 10% w/v suspension of human fecal material in PBS was incubated in awater bath at 80 degrees Celsius for 30 minutes. Glycerol was added to afinal concentration of 15% and then the enriched spore containingmaterial was stored at −80 degrees Celsius.

Example 4. Spore Purification from Alcohol Treatment and ThermalTreatment of Fecal Material

A 10% w/v suspension of human feces in PBS was mixed with absoluteethanol in a 1:1 ratio and vortexed to mix for 1 minute. The suspensionwas incubated in a water bath under aerobic conditions at 37 degreesCelsius for 1 hour. After incubation the suspension was centrifuged at13,000 rpm for 5 minutes to pellet spores. The supernatant was discardedand the pellet was resuspended in equal volume PBS. The ethanol treatedspore population was then incubated in a water bath at 80 degreesCelsius for 30 minutes. Glycerol was added to a final concentration of15% and the purified spore fraction was stored at −80 C.

Example 5. Spore Purification from Detergent Treatment of Fecal Material

A 10% w/v suspension of human feces in PBS is prepared to contain afinal concentration of 0.5 to 2% Triton X-100. After shaking incubationfor 30 minutes at 25 to 37 degrees Celsius, the sample is centrifuged at1000 g for 5-10 minutes to pellet particulate matter and large cells.The bacterial spores are recovered in the supernatant fraction, wherethe purified spore population is optionally further treated, such as inExample 4. Without being bound by theory, detergent addition to thefecal mixture produces better spore populations, at least in part byenhancing separation of the spores from particulates thereby resultingin higher yields of spores.

Example 6. Spore Purification by Chromatographic Separation of FecalMaterial

A spore-enriched population such as obtained from Examples 1-5 above, ismixed with NaCl to a final concentration of 4M total salt and contactedwith octyl Sepharose 4 Fast Flow to bind the hydrophobic spore fraction.The resin is washed with 4M NaCl to remove less hydrophobic components,and the spores are eluted with distilled water, and the desired enrichedspore fraction is collected via UV absorbance.

Example 7. Spore Purification by Filtration of Fecal Material

A spore-enriched population such as obtained from Examples 1-6 above isdiluted 1:10 with PBS, and placed in the reservoir vessel of atangential flow microfiltration system. A 0.2 um pore size mixedcellulose ester hydrophilic tangential flow filter is connected to thereservoir such as by a tubing loop. The diluted spore preparation isrecirculated through the loop by pumping, and the pressure gradientacross the walls of the microfilter forces the supernatant liquidthrough the filter pores. By appropriate selection of the filter poresize the desired bacterial spores are retained, while smallercontaminants such as cellular debris, and other contaminants in fecessuch as bacteriophage pass through the filter. Fresh PBS buffer is addedto the reservoir periodically to enhance the washout of thecontaminants. At the end of the diafiltration, the spores areconcentrated approximately ten-fold to the original concentration. Thepurified spores are collected from the reservoir and stored as providedherein.

Example 8. Characterization of Purified Spore Populations

Counts of viable spores are determined by performing 10 fold serialdilutions in PBS and plating to Brucella Blood Agar Petri plates orapplicable solid media. Plates are incubated at 37 degrees Celsius for 2days. Colonies are counted from a dilution plate with 50-400 coloniesand used to back-calculate the number of viable spores in thepopulation. The ability to germinate into vegetative bacteria is alsodemonstrated. Visual counts are determined by phase contrast microscopy.A spore preparation is either diluted in PBS or concentrated bycentrifugation, and a 5 microliter aliquot is placed into a PetroffHauser counting chamber for visualization at 400× magnification. Sporesare counted within ten 0.05 mm×0.05 mm grids and an average spore countper grid is determined and used to calculate a spore count per ml ofpreparation. Lipopolysaccharide (LPS) reduction in purified sporepopulations is measured using a Limulus amebocyte lysate (LAL) assaysuch as the commercially available ToxinSensor™ Chromogenic LALEndotoxin Assay Kit (GenScript, Piscataway, N.J.) or other standardmethods known to those skilled in the art.

Example 9. Determination of Bacterial Pathogens in Purified SporePopulations

Bacterial pathogens present in a purified spore population aredetermined by qPCR using specific oligonucleotide primers as follows.

Standard Curve Preparation. The standard curve is generated from wellscontaining the pathogen of interest at a known concentration orsimultaneously quantified by selective spot plating. Serial dilutions ofduplicate cultures are performed in sterile phosphate-buffered saline.Genomic DNA is then extracted from the standard curve samples along withthe other samples.

Genomic DNA Extraction.

Genomic DNA may be extracted from 100 μl of fecal samples, fecal-derivedsamples, or purified spore preparations using the Mo Bio Powersoil®-htp96 Well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.)according to the manufacturer's instructions with two exceptions: thebeadbeating is performed for 2×4:40 minutes using a BioSpecMini-Beadbeater-96 (BioSpec Products, Bartlesville, Okla.) and the DNAis eluted in 50 μl of Solution C6. Alternatively the genomic DNA couldbe isolated using the Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), the Sigma-Aldrich Extract-N-Amp™ PlantPCR Kit, the QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, Calif.)according to the manufacturer's instructions.

[qPCR Composition and Conditions.

The qPCR reaction to detect C. difficile contains 1× HotMasterMix(5PRIME, Gaithersburg, Md.), 900 nM of Wr-tcdB-F(AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG, (SEQ ID NO: 2040) IDT, Coralville,Iowa), 900 nM of Wr-tcdB-R (CATGCTTTTTTAGTTTCTGGATTGAA, (SEQ ID NO:2041) IDT, Coralville, Iowa), 250 nM of We-tcdB-P(6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQ ID NO: 2042), LifeTechnologies, Grand Island, N.Y.), and PCR Water (Mo Bio Laboratories,Carlsbad, Calif.) to 18 μl (Primers adaped from: Wroblewski, D. et al.Rapid Molecular Characterization of Clostridium difficile and Assessmentof Populations of C. difficile in Stool Specimens. Journal of ClinicalMicrobiology 47:2142-2148 (2009)). This reaction mixture is aliquoted towells of a MicroAmp® Fast Optical 96-well Reaction Plate with Barcode(0.1 mL) (Life Technologies, Grand Island, N.Y.). To this reactionmixture, 2 μl of extracted genomic DNA is added. The qPCR is performedon a BioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-TimeSystem (BioRad, Hercules, Calif.). The thermocycling conditions are 95°C. for 2 minutes followed by 45 cycles of 95° C. for 3 seconds, 60° C.for 30 seconds, and fluorescent readings of the FAM and ROX channels.Other bacterial pathogens can be detected by using primers and a probespecific for the pathogen of interest.

Data Analysis.

The Cq value for each well on the FAM channel is determined by the CFXManager™ Software Version 2.1. The log 10 (cfu/ml) of each experimentalsample is calculated by inputting a given sample's Cq value into linearregression model generated from the standard curve comparing the Cqvalues of the standard curve wells to the known log 10 (cfu/ml) of thosesamples.

[Viral pathogens present in a purified spore population are determinedby qPCR as described herein and otherwise known in the art.

Example 10: Species Identification

The identity of the spore-forming species which grew up from a complexfraction can be determined in multiple ways. First, individual coloniescan be picked into liquid media in a 96 well format, grown up and savedas 15% glycerol stocks at −80 C. Aliquots of the cultures can be placedinto cell lysis buffer and colony PCR methods can be used to amplify andsequence the 16S rDNA gene (Example 2). Alternatively, colonies may bestreaked to purity in several passages on solid media. Well separatedcolonies are streaked onto the fresh plates of the same kind andincubated for 48-72 hours at 37 C. The process is repeated multipletimes in order to ensure purity. Pure cultures can be analyzed byphenotypic- or sequence-based methods, including 16S rDNA amplificationand sequencing as described in Examples 11 & 12. Sequencecharacterization of pure isolates or mixed communities e.g. platescrapes and spore fractions can also include whole genome shotgunsequencing. The latter is valuable to determine the presence of genesassociated with sporulation, antibiotic resistance, pathogenicity, andvirulence. Colonies can also be scraped from plates en masse andsequenced using a massively parallel sequencing method as described inExamples 11 & 12 such that individual 16S signatures can be identifiedin a complex mixture. Optionally, the sample can be sequenced prior togermination (if appropriate DNA isolation procedures are used to lsyeand release the DNA from spores) in order to compare the diversity ofgerminable species with the total number of species in a spore sample.As an alternative or complementary approach to 16S analysis,MALDI-TOF-mass spec can also be used for species identification (asreviewed in Anaerobe 22:123).

Example 11: 16s Sequencing to Determine Operational Taxonomic Unit (OTU)

Method for Determining 16S Sequence

OTUs may be defined either by full 16S sequencing of the rRNA gene, bysequencing of a specific hypervariable region of this gene (i.e. V1, V2,V3, V4, V5, V6, V7, V8, or V9), or by sequencing of any combination ofhypervariable regions from this gene (e.g. V1-3 or V3-5). The bacterial16S rDNA is approximately 1500 nucleotides in length and is used inreconstructing the evolutionary relationships and sequence similarity ofone bacterial isolate to another using phylogenetic approaches. 16Ssequences are used for phylogenetic reconstruction as they are ingeneral highly conserved, but contain specific hypervariable regionsthat harbor sufficient nucleotide diversity to differentiate genera andspecies of most microbes.

Using well known techniques, in order to determine the full 16S sequenceor the sequence of any hypervariable region of the 16S sequence, genomicDNA is extracted from a bacterial sample, the 16S rDNA (full region orspecific hypervariable regions) amplified using polymerase chainreaction (PCR), the PCR products cleaned, and nucleotide sequencesdelineated to determine the genetic composition of 16S gene or subdomainof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morehypervariable regions are used, such as the V4 region, the sequencingmay be, but is not limited to being, performed using the Sanger methodor using a next-generation sequencing method, such as an Illumina(sequencing by synthesis) method using barcoded primers allowing formultiplex reactions.

In addition to the 16S rRNA gene, one may define an OTU by sequencing aselected set of genes that are known to be marker genes for a givenspecies or taxonomic group of OTUs. These genes may alternatively beassayed using a PCR-based screening strategy. As example, variousstrains of pathogenic Escherichia coli can be distinguished using DNAsfrom the genes that encode heat-labile (LTI, LTIIa, and LTIIb) andheat-stable (STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1,VT2, and VT2e, respectively), cytotoxic necrotizing factors (CNF1 andCNF2), attaching and effacing mechanisms (eaeA), enteroaggregativemechanisms (Eagg), and enteroinvasive mechanisms (Einv). The optimalgenes to utilize for taxonomic assignment of OTUs by use of marker geneswill be familiar to one with ordinary skill of the art of sequence basedtaxonomic identification.

Genomic DNA Extraction

Genomic DNA is extracted from pure microbial cultures using a hotalkaline lysis method. 1 μl of microbial culture is added to 9 μl ofLysis Buffer (25 mM NaOH, 0.2 mM EDTA) and the mixture is incubated at95° C. for 30 minutes. Subsequently, the samples are cooled to 4° C. andneutralized by the addition of 10 μl of Neutralization Buffer (40 mMTris-HCl) and then diluted 10-fold in Elution Buffer (10 mM Tris-HCl).Alternatively, genomic DNA is extracted from pure microbial culturesusing commercially available kits such as the Mo Bio Ultraclean®Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, Calif.) orby standard methods known to those skilled in the art.

Amplification of 16S Sequences for Downstream Sanger Sequencing

To amplify bacterial 16S rDNA (FIG. 1A), 2 μl of extracted gDNA is addedto a 20 μl final volume PCR reaction. For full-length 16 sequencing thePCR reaction also contains 1× HotMasterMix (5FRIME, Gaithersburg, Md.),250 nM of 27f (AGRGTTTGATCMTGGCTCAG (SEQ ID NO: 2033), IDT, Coralville,Iowa), and 250 nM of 1492r (TACGGYTACCTTGTTAYGACTT (SEQ ID NO: 2034),IDT, Coralville, Iowa), with PCR Water (Mo Bio Laboratories, Carlsbad,Calif.) for the balance of the volume. Alternatively, other universalbacterial primers or thermostable polymerases known to those skilled inthe art are used. For example primers are available to those skilled inthe art for the sequencing of the “V1-V9 regions” of the 16S rRNA (FIG.1A). These regions refer to the first through ninth hypervariableregions of the 16S rRNA gene that are used for genetic typing ofbacterial samples. These regions in bacteria are defined by nucleotides69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173,1243-1294 and 1435-1465 respectively using numbering based on the E.coli system of nomenclature. Brosius et al., Complete nucleotidesequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS75(10):4801-4805 (1978). In some embodiments, at least one of the V1,V2, V3, V4, V5, V6, V7, V8, and V9 regions are used to characterize anOTU. In one embodiment, the V1, V2, and V3 regions are used tocharacterize an OTU. In another embodiment, the V3, V4, and V5 regionsare used to characterize an OTU. In another embodiment, the V4 region isused to characterize an OTU. A person of ordinary skill in the art canidentify the specific hypervariable regions of a candidate 16S rRNA (inFIG. 1A) by comparing the candidate sequence in question to thereference sequence (FIG. 1B) and identifying the hypervariable regionsbased on similarity to the reference hypervariable regions.

The PCR is performed on commercially available thermocyclers such as aBioRad MyCycler™ Thermal Cycler (BioRad, Hercules, Calif.). Thereactions are run at 94° C. for 2 minutes followed by 30 cycles of 94°C. for 30 seconds, 51° C. for 30 seconds, and 68° C. for 1 minute 30seconds, followed by a 7 minute extension at 72° C. and an indefinitehold at 4° C. Following PCR, gel electrophoresis of a portion of thereaction products is used to confirm successful amplification of a ˜1.5kb product.

To remove nucleotides and oligonucleotides from the PCR products, 2 μlof HT ExoSap-IT (Affymetrix, Santa Clara, Calif.) is added to 5 μl ofPCR product followed by a 15 minute incubation at 37° C. and then a 15minute inactivation at 80° C.

Amplification of 16S Sequences for Downstream Characterization byMassively Parallel Sequencing Technologies

Amplification performed for downstream sequencing by short readtechnologies such as Illumina require amplification using primers knownto those skilled in the art that additionally include a sequence-basedbarcoded tag. As example, to amplify the 16s hypervariable region V4region of bacterial 16S rDNA, 2 μl of extracted gDNA is added to a 20 μlfinal volume PCR reaction. The PCR reaction also contains 1×HotMasterMix (5PRIME, Gaithersburg, Md.), 200 nM of V4_515f_adapt(AATGATACGGCGACCACCGAGATCTACACTATGGTAATTGTGTGCCAGCMGCC GCGGTAA (SEQ IDNO: 2035), IDT, Coralville, Iowa), and 200 nM of barcoded 806rbc(CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 2036)_12bpGolayBarcode_AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 2037), IDT,Coralville, Iowa), with PCR Water (Mo Bio Laboratories, Carlsbad,Calif.) for the balance of the volume. These primers incorporatebarcoded adapters for Illumina sequencing by synthesis. Optionally,identical replicate, triplicate, or quadruplicate reactions may beperformed. Alternatively other universal bacterial primers orthermostable polymerases known to those skilled in the art are used toobtain different amplification and sequencing error rates as well asresults on alternative sequencing technologies.

The PCR amplification is performed on commercially availablethermocyclers such as a BioRad MyCycler™ Thermal Cycler (BioRad,Hercules, Calif.). The reactions are run at 94° C. for 3 minutesfollowed by 25 cycles of 94° C. for 45 seconds, 50° C. for 1 minute, and72° C. for 1 minute 30 seconds, followed by a 10 minute extension at 72°C. and a indefinite hold at 4° C. Following PCR, gel electrophoresis ofa portion of the reaction products is used to confirm successfulamplification of a ˜1.5 kb product. PCR cleanup is performed asspecified in the previous example.

Sanger Sequencing of Target Amplicons from Pure Homogeneous Samples

To detect nucleic acids for each sample, two sequencing reactions areperformed to generate a forward and reverse sequencing read. Forfull-length 16s sequencing primers 27f and 1492r are used. 40 ng ofExoSap-IT-cleaned PCR products are mixed with 25 pmol of sequencingprimer and Mo Bio Molecular Biology Grade Water (Mo Bio Laboratories,Carlsbad, Calif.) to 15 μl total volume. This reaction is submitted to acommercial sequencing organization such as Genewiz (South Plainfield,N.J.) for Sanger sequencing.

Massively Parallel Sequencing of Target Amplicons from HeterogeneousSamples

DNA Quantification & Library Construction.

The cleaned PCR amplification products are quantified using theQuant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies, Grand Island,N.Y.) according to the manufacturer's instructions. Followingquantification, the barcoded cleaned PCR products are combined such thateach distinct PCR product is at an equimolar ratio to create a preparedIllumina library.

Nucleic Acid Detection.

The prepared library is sequenced on Illumina HiSeq or MiSeq sequencers(Illumina, San Diego, Calif.) with cluster generation, templatehybridization, iso-thermal amplification, linearization, blocking anddenaturization and hybridization of the sequencing primers performedaccording to the manufacturer's instructions. 16SV4SeqFw(TATGGTAATTGTGTGCCAGCMGCCGCGGTAA (SEQ ID NO: 2038)), 16SV4SeqRev(AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT (SEQ ID NO: 2037)), and 16SV4Index(ATTAGAWACCCBDGTAGTCCGGCTGACTGACT (SEQ ID NO: 2039)) (IDT, Coralville,Iowa) are used for sequencing. Other sequencing technologies can be usedsuch as but not limited to 454, Pacific Biosciences, Helicos, IonTorrent, and Nanopore using protocols that are standard to someoneskilled in the art of genomic sequencing.

Example 12: Sequence Read Annotation

Primary Read Annotation

Nucleic acid sequences are analyzed and annotations are to definetaxonomic assignments using sequence similarity and phylogeneticplacement methods or a combination of the two strategies. A similarapproach can be used to annotate protein names, transcription factornames, and any other classification schema for nucleic acid sequences.Sequence similarity based methods include those familiar to individualsskilled in the art including, but not limited to BLAST, BLASTx, tBLASTn,tBLASTx, RDP-classifier, DNAclust, and various implementations of thesealgorithms such as Qiime or Mothur. These methods rely on mapping asequence read to a reference database and selecting the match with thebest score and e-value. Common databases include, but are not limited tothe Human Microbiome Project, NCBI non-redundant database, Greengenes,RDP, and Silva. Phylogenetic methods can be used in combination withsequence similarity methods to improve the calling accuracy of anannotation or taxonomic assignment. Here tree topologies and nodalstructure are used to refine the resolution of the analysis. In thisapproach we analyze nucleic acid sequences using one of numeroussequence similarity approaches and leverage phylogenetic methods thatare well known to those skilled in the art, including but not limited tomaximum likelihood phylogenetic reconstruction (see e.g. Liu K, Linder CR, and Warnow T. 2011. RAxML and FastTree: Comparing Two Methods forLarge-Scale Maximum Likelihood Phylogeny Estimation. PLoS ONE 6: e27731.McGuire G, Denham M C, and Balding D J. 2001. Models of sequenceevolution for DNA sequences containing gaps. Mol. Biol. Evol 18:481-490. Wróbel B. 2008. Statistical measures of uncertainty forbranches in phylogenetic trees inferred from molecular sequences byusing model-based methods. J. Appl. Genet. 49: 49-67.) Sequence readsare placed into a reference phylogeny comprised of appropriate referencesequences. Annotations are made based on the placement of the read inthe phylogenetic tree. The certainty or significance of the OTUannotation is defined based on the OTU's sequence similarity to areference nucleic acid sequence and the proximity of the OTU sequencerelative to one or more reference sequences in the phylogeny. As anexample, the specificity of a taxonomic assignment is defined withconfidence at the the level of Family, Genus, Species, or Strain withthe confidence determined based on the position of bootstrap supportedbranches in the reference phylogenetic tree relative to the placement ofthe OTU sequence being interrogated.

Clade Assignments

The ability of 16S-V4 OTU identification to assign an OTU as a specificspecies depends in part on the resolving power of the 16S-V4 region ofthe 16S gene for a particular species or group of species. Both thedensity of available reference 16S sequences for different regions ofthe tree as well as the inherent variability in the 16S gene betweendifferent species will determine the definitiveness of a taxonomicannotation. Given the topological nature of a phylogenetic tree and thefact that tree represents hierarchical relationships of OTUs to oneanother based on their sequence similarity and an underlyingevolutionary model, taxonomic annotations of a read can be rolled up toa higher level using a clade-based assignment procedure (Table 1). Usingthis approach, clades are defined based on the topology of aphylogenetic tree that is constructed from full-length 16S sequencesusing maximum likelihood or other phylogenetic models familiar toindividuals with ordinary skill in the art of phylogenetics. Clades areconstructed to ensure that all OTUs in a given clade are: (i) within aspecified number of bootstrap supported nodes from one another(generally, 1-5 bootstraps), and (ii) within a 5% genetic similarity.OTUs that are within the same clade can be distinguished as geneticallyand phylogenetically distinct from OTUs in a different clade based on16S-V4 sequence data. OTUs falling within the same clade areevolutionarily closely related and may or may not be distinguishablefrom one another using 16S-V4 sequence data. The power of clade basedanalysis is that members of the same clade, due to their evolutionaryrelatedness, are likely to play similar functional roles in a microbialecology such as that found in the human gut. Compositions substitutingone species with another from the same clade are likely to haveconserved ecological function and therefore are useful in the presentinvention.

Notably, 16S sequences of isolates of a given OTU are phylogeneticallyplaced within their respective clades, sometimes in conflict with themicrobiological-based assignment of species and genus that may havepreceded 16S-based assignment. Discrepancies between taxonomicassignment based on microbiological characteristics versus geneticsequencing are known to exist from the literature.

Example 13: Germinating Spores

Germinating a spore fraction increases the number of viable spores thatwill grow on various media types. To germinate a population of spores,the sample is moved to the anaerobic chamber, resuspended in prereducedPBS, mixed and incubated for 1 hour at 37 C to allow for germination.Germinants can include amino-acids (e.g., alanine, glycine), sugars(e.g., fructose), nucleosides (e.g., inosine), bile salts (e.g., cholateand taurocholate), metal cations (e.g., Mg2+, Ca2+), fatty acids, andlong-chain alkyl amines (e.g., dodecylamine, Germination of bacterialspores with alkyl primary amines” J. Bacteriology, 1961.). Mixtures ofthese or more complex natural mixtures, such as rumen fluid or Oxgall,can be used to induce germination. Oxgall is dehydrated bovine bilecomposed of fatty acids, bile acids, inorganic salts, sulfates, bilepigments, cholesterol, mucin, lecithin, glycuronic acids, porphyrins,and urea. The germination can also be performed in a growth medium likeprereduced BHIS/oxgall germination medium, in which BHIS (Brain heartinfusion powder (37 g/L), yeast extract (5 g/L), L-cysteine HCl (1 g/L))provides peptides, amino acids, inorganic ions and sugars in the complexBHI and yeast extract mixtures and Oxgall provides additional bile acidgerminants.

In addition, pressure may be used to germinate spores. The selection ofgerminants can vary with the microbe being sought. Different speciesrequire different germinants and different isolates of the same speciescan require different germinants for optimal germination. Finally, it isimportant to dilute the mixture prior to plating because some germinantsare inhibitory to growth of the vegetative-state microorganisms. Forinstance, it has been shown that alkyl amines must be neutralized withanionic lipophiles in order to promote optimal growth. Bile acids canalso inhibit growth of some organisms despite promoting theirgermination, and must be diluted away prior to plating for viable cells.

For example, BHIS/oxgall solution is used as a germinant and contains0.5×BHIS medium with 0.25% oxgall (dehydrated bovine bile) where 1×BHISmedium contains the following per L of solution: 6 g Brain HeartInfusion from solids, 7 g peptic digest of animal tissue, 14.5 g ofpancreatic digest of casein, 5 g of yeast extract, 5 g sodium chloride,2 g glucose, 2.5 g disodium phosphate, and 1 g cysteine. Additionally,Ca-DPA is a germinant and contains 40 mM CaCl2, and 40 mM dipicolinicacid (DPA). Rumen fluid (Bar Diamond, Inc.) is also a germinant.Simulated gastric fluid (Ricca Chemical) is a germinant and is 0.2%(w/v) Sodium Chloride in 0.7% (v/v) Hydrochloric Acid. Mucin medium is agerminant and prepared by adding the following items to 1 L of distilledsterile water: 0.4 g KH₂PO₄, 0.53 g Na₂HPO₄, 0.3 g NH₄Cl, 0.3 g NaCl,0.1 g MgCl₂×6H₂O, 0.11 g CaCl₂, 1 ml alkaline trace element solution, 1ml acid trace element solution, 1 ml vitamin solution, 0.5 mg resazurin,4 g NaHCO₃, 0.25 g Na₂S×9 H₂O. The trace element and vitamin solutionsprepared as described previously (Stams et al., 1993). All compoundswere autoclaved, except the vitamins, which were filter-sterilized. Thebasal medium was supplemented with 0.7% (v/v) clarified, sterile rumenfluid and 0.25% (v/v) commercial hog gastric mucin (Type Sigma),purified by ethanol precipitation as described previously (Miller &Hoskins, 1981). This medium is referred herein as mucin medium.

Fetal Bovine Serum (Gibco) can be used as a germinant and contains 5%FBS heat inactivated, in Phosphate Buffered Saline (PBS, FisherScientific) containing 0.137M Sodium Chloride, 0.0027M PotassiumChloride, 0.0119M Phosphate Buffer. Thioglycollate is a germinant asdescribed previously (Kamiya et al Journal of Medical Microbiology 1989)and contains 0.25M (pH10) sodium thioglycollate. Dodecylamine solutioncontaining 1 mM dodecylamine in PBS is a germinant. A sugar solution canbe used as a germinant and contains 0.2% fructose, 0.2% glucose, and0.2% mannitol. Amino acid solution can also be used as a germinant andcontains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mMproline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine. Agerminant mixture referred to herein as Germix 3 can be a germinant andcontains 5 mM alanine, 1 mM arginine, 1 mM histidine, 1 mM lysine, 1 mMproline, 1 mM asparagine, 1 mM aspartic acid, 1 mM phenylalanine, 0.2%taurocholate, 0.2% fructose, 0.2% mannitol, 0.2% glucose, 1 mM inosine,2.5 mM Ca-DPA, and 5 mM KCl. BHIS medium+DPA is a germinant mixture andcontains BHIS medium and 2 mM Ca-DPA. Escherichia coli spent mediumsupernatant referred to herein as EcSN is a germinant and is prepared bygrowing E. coli MG1655 in SweetB/Fos inulin medium anaerobically for 48hr, spinning down cells at 20,000 rcf for 20 minutes, collecting thesupernatant and heating to 60 C for 40 min. Finally, the solution isfilter sterilized and used as a germinant solution.

Example 14: Selection of Media for Growth

It is important to select appropriate media to support growth, includingpreferred carbon sources. For example, some organisms prefer complexsugars such as cellobiose over simple sugars. Examples of media used inthe isolation of sporulating organisms include EYA, BHI, BHIS, and GAM(see below for complete names and references). Multiple dilutions areplated out to ensure that some plates will have well isolated colonieson them for analysis, or alternatively plates with dense colonies mayscraped and suspended in PBS to generate a mixed diverse community.

Plates are incubated anaerobically or aerobically at 37 C for 48-72 ormore hours, targeting anaerobic or aerobic spore formers, respectively.

Solid plate media include:

-   -   Gifu Anaerobic Medium (GAM, Nissui) without dextrose        supplemented with fructooligosaccharides/inulin (0.4%), mannitol        (0.4%), inulin (0.4%), or fructose (0.4%), or a combination        thereof.    -   Sweet GAM [Gifu Anaerobic Medium (GAM, Nissui)] modified,        supplemented with glucose, cellobiose, maltose, L-arabinose,        fructose, fructooligosaccharides/inulin, mannitol and sodium        lactate)    -   Brucella Blood Agar (BBA, Atlas, Handbook of Microbiological        Media, 4th ed, ASM Press, 2010)    -   PEA sheep blood (Anaerobe Systems; 5% Sheep Blood Agar with        Phenylethyl Alcohol)    -   Egg Yolk Agar (EYA) (Atlas, Handbook of Microbiological Media,        4th ed, ASM Press, 2010)    -   Sulfite polymyxin milk agar (Mevissen-Verhage et al., J. Clin.        Microbiol. 25:285-289 (1987))    -   Mucin agar (Derrien et al., IJSEM 54: 1469-1476 (2004))    -   Polygalacturonate agar (Jensen & Canale-Parola, Appl. Environ.        Microbiol. 52:880-997 (1986))    -   M2GSC (Atlas, Handbook of Microbiological Media, 4th ed, ASM        Press, 2010)    -   M2 agar (Atlas, Handbook of Microbiological Media, 4th ed, ASM        Press, 2010) supplemented with starch (1%), mannitol (0.4%),        lactate (1.5 g/L) or lactose (0.4%)    -   Sweet B—Brain Heart Infusion agar (Atlas, Handbook of        Microbiological Media, 4th ed, ASM Press, 2010) supplemented        with yeast extract (0.5%), hemin, cysteine (0.1%), maltose        (0.1%), cellobiose (0.1%), soluble starch (sigma, 1%), MOPS (50        mM, pH 7).    -   PY-salicin (peptone-yeast extract agar supplemented with        salicin) (Atlas, Handbook of Microbiological Media, 4th ed, ASM        Press, 2010).    -   Modified Brain Heart Infusion (M-BHI) [[sweet and sour]]        contains the following per L: 37.5 g Brain Heart Infusion powder        (Remel), 5 g yeast extract, 2.2 g meat extract, 1.2 g liver        extract, 1 g cystein HCl, 0.3 g sodium thioglycolate, 10 mg        hemin, 2 g soluble starch, 2 g FOS/Inulin, 1 g cellobiose, 1 g        L-arabinose, 1 g mannitol, 1 Na-lactate, 1 mL Tween 80, 0.6 g        MgSO4×7H2O, 0.6 g CaCl2, 6 g (NH4)2SO4, 3 g KH2PO4, 0.5 g        K2HPO4, 33 mM Acetic acid, 9 mM propionic acid, 1 mM Isobutyric        acid, 1 mM isovaleric acid, 15 g agar, and after autoclaving add        50 mL of 8% NaHCO₃ solution and 50 mL 1M MOPS-KOH (pH 7).    -   Noack-Blaut Eubacterium agar (See Noack et al. J. Nutr. (1998)        128:1385-1391)    -   BHIS az1/ge2—BHIS az/ge agar (Reeves et. al. Infect. Immun.        80:3786-3794 (2012)) [Brain Heart Infusion agar (Atlas, Handbook        of Microbiological Media, 4th ed, ASM Press, 2010) supplemented        with yeast extract 0.5%, cysteine 0.1%, 0.1% cellobiose, 0.1%        inulin, 0.1% maltose, aztreonam 1 mg/L, gentamycin 2 mg/L]    -   BHIS ClnM az1/ge2—BHIS ClnM [Brain Heart Infusion agar (Atlas,        Handbook of Microbiological Media, 4th ed, ASM Press, 2010)        supplemented with yeast extract 0.5%, cysteine 0.1%, 0.1%        cellobiose, 0.1% inulin, 0.1% maltose, aztreonam 1 mg/L,        gentamycin 2 mg/L]

Example 15: The Purification and Isolation of a Spore Forming Fractionfrom Feces

To purify and selectively isolate efficacious spores from fecal materiala donation is first blended with saline using a homogenization device(e.g., laboratory blender) to produce a 20% slurry (w/v). 100% ethanolis added for an inactivation treatment that lasts 10 seconds to 1 hour.The final alcohol concentration can range from 30-90%, preferably50-70%. High speed centrifugation (3200 rcf for 10 min) is performed toremove solvent and the pellet is retained and washed. Subsequently, oncethe washed pellet is resuspended, a low speed centrifugation step (200rcf for 4 min) is performed to remove large particulate vegetativematter and the supernatant containing the spores is retained. High speedcentrifugation (3200 rcf for 10 min) is performed on the supernatant toconcentrate the spore material. The pellet is then washed andresuspended to generate a 20% slurry. This is the ethanol treated sporepreparation. The concentrated slurry is then separated with a densitybased gradient e.g. a CsCl gradient, sucrose gradient or combination ofthe two generating a ethanol treated, gradient-purified sporepreparation. For example, a CsCl gradient is performed by loading a 20%volume of spore suspension on top a 80% volume of a stepwise CsClgradient (w/v) containing the steps of 64%, 50%, 40% CsCl (w/v) andcentrifuging for 20 min at 3200 rcf. The spore fraction is then run on asucrose step gradient with steps of 67%, 50%, 40%, and 30% (w/v). Whencentrifuged in a swinging bucket rotor for 10 min at 3200 rcf. Thespores run roughly in the 30% and 40% sucrose fractions. The lower sporefraction (FIG. 2) is then removed and washed to produce a concentratedethanol treated, gradient-purified spore preparation. Taking advantageof the refractive properties of spores observed by phase contrastmicroscopy (spores are bright and refractive while germinated spores andvegetative cells are dark) one can see an enrichment of the sporefraction from a fecal bacterial cell suspension (FIG. 3, left) comparedto an ethanol treated, CsCl gradient purified, spore preparation (FIG.3, center), and to an ethanol treated, CsCl gradient purified, sucrosegradient purified, spore preparation (FIG. 3, right).

Furthermore, growth of spores after treatment with a germinant can alsobe used to quantify a viable spore population. Briefly, samples wereincubated with a germinant (Oxgall, 0.25% for up to 1 hour), diluted andplated anaerobically on BBA (Brucella Blood Agar) or similar media (e.g.see Examples 4 and 5). Individual colonies were picked and DNA isolatedfor full-length 16S sequencing to identify the species composition (e.g.see examples 2 and 3). Analysis revealed that 22 species were observedin total (Table 2) with a vast majority present in both the materialpurified with the gradient and without the gradient, indicating no orinconsequential shift in the ecology as a result of gradientpurification. Spore yield calculations demonstrate an efficient recoveryof 38% of the spores from the initial fecal material as measured bygermination and plating of spores on BBA or measuring DPA count in thesample.

Example 16: Bacterial Compositions Prevent C. difficile Infection in aMouse Model

To test the therapeutic potential of the bacterial compositions aprophylactic mouse model of C. difficile infection (model based on Chen,et al., A mouse model of Clostridium difficile associated disease,Gastroenterology 135(6):1984-1992) was used. Two cages of five mice eachwere tested for each arm of the experiment. All mice received anantibiotic cocktail consisting of 10% glucose, kanamycin (0.5 mg/ml),gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) andVancomycin (0.056 mg/ml) in their drinking water on days −14 through −5and a dose of 10 mg/kg Clindamycin by oral gavage on day −3. On day −1,they received either the test article or vehicle control via oralgavage. On day 0 they were challenged by administration of approximately4.5 log 10 cfu of C. difficile (ATCC 43255) via oral gavage. Optionallya positive control group received vancomycin from day −1 through day 3in addition to the antibiotic protocol and C. difficile challengespecified above. Feces were collected from the cages for analysis ofbacterial carriage, mortality was assessed every day from day 0 to day 6and the weight and subsequent weight change of the animal was assessedwith weight loss being associated with C. difficile infection. Mortalityand reduced weight loss of the test article compared to the vehicle wereused to assess the success of the test article. Additionally, a C.difficile symptom scoring was performed each day from day −1 through day6. Clinical Score was based on a 0-4 scale by combining scores forAppearance (0-2 pts based on normal, hunched, piloerection, orlethargic), and Clinical Signs (0-2 points based on normal, wet tail,cold-to-the-touch, or isolation from other animals).

In a naive control arm, animals were challenged with C. difficile. Inthe vancomycin positive control arm animals were dosed with C. difficileand treated with vancomycin from day −1 through day 3. The negativecontrol was gavaged with PBS alone and no bacteria. The test arms of theexperiment tested 1×, 0.1×, 0.01× dilutions derived from a single donorpreparation of ethanol treated spores (e.g. see example 6) or the heattreated feces prepared by treating a 20% slurry for 30 min at 80 C.Dosing for CFU counts was determined from the final ethanol treatedspores and dilutions of total spores were administered at 1×, 0.1×,0.01× of the spore mixture for the ethanol treated fraction and a 1×dose for the heat treated fraction.

Weight loss and mortality were assessed on day 3. The negative control,treated with C. difficile only, exhibits 20% mortality and weight losson Day 3, while the positive control of 10% human fecal suspensiondisplays no mortality or weight loss on Day 3 (Table 3). EtOH-treatedfeces prevents mortality and weight loss at three dilutions, while theheat-treated fraction was protective at the only dose tested. These dataindicate that the spore fraction is efficacious in preventing C.difficile infection in the mouse.

Example 17: The Prophylactic and Relapse Prevention Hamster Models

Previous studies with hamsters using toxigenic and nontoxigenic strainsof C. difficile demonstrated the utility of the hamster model inexamining relapse post antibiotic treatment and the effects ofprophylaxis treatments with cecal flora in C. difficile infection(Wilson et al. 1981, Wilson et al. 1983, Borriello et al. 1985) and morebroadly gastrointestinal infectious disease. To demonstrate prophylacticuse of a test article to ameliorate C. difficile infection, thefollowing hamster model is used. In a prophylactic model, Clindamycin(10 mg/kg s.c.) is given on day −5, the test article or control isadministered on day −3, and C. difficile challenge occurs on day 0. Inthe positive control arm, vancomycin is then administered on day 1-5(and vehicle control is delivered on day −3). Feces are collected on day−5, −4, −1, 1, 3, 5, 7, 9 and fecal samples are assessed for pathogencarriage and reduction by microbiological methods, 16S sequencingapproaches or other methods utilized by one skilled in the art.Mortality is assessed throughout the experiment through 21 days post C.difficile challenge. The percentage survival curves show that ethanoltreated spores and ethanol treated, gradient-purified spores betterprotect the hamsters compared to the Vancomycin control, and vehiclecontrol.

See FIG. 4: Prophylaxis Model with the Ethanol Treated Spore Preparationand the Ethanol Treated, Gradient-Purified Spore Preparation.

In the relapse prevention model, hamsters are challenged with toxigenicC. difficile strains on day 0, and treated with clindamycin by oralgavage on day 1, and vancomycin dosing day 2-6. Test or controltreatment was then administered on day 7, 8, and 9. The groups ofhamsters for each arm consist of 8 hamsters per group. Fecal material iscollected on day −1, 1, 3, 5, 7, 10 and 13 and hamster mortality isassessed throughout. Survival curves are used to assess the success ofthe test article e.g. ethanol treated or ethanol treated, gradientpurified spores versus the control treatment in preventing hamsterdeath. The survival curves demonstrate maximum efficacy for the ethanoltreated, gradient-purified spores followed by the ethanol treatedspores. Both treatments improved survival percentage over vancomycintreatment alone.

See FIG. 5: Relapse Prevention Model with Ethanol Treated Spores andEthanol Treated, Gradient Purified Spores

Example 18: Clinical Treatment of Recurrent C. difficile in Patients

To assess the efficacy of a test article (e.g., ethanol treated sporepreparations, see Example 15) to treat recurrent C. difficile in humanpatients, the following procedure was performed to take feces from ahealthy donor, inactivate via the ethanol treated spore preparationprotocol described below, and treat recurrent C. difficile in patientspresenting with this indication. Non-related donors were screened forgeneral health history for absence of chronic medical conditions(including inflammatory bowel disease; irritable bowel syndrome; Celiacdisease; or any history of gastrointestinal malignancy or polyposis),absence of risk factors for transmissible infections, antibiotic non-usein the previous 6 months, and negative results in laboratory assays forblood-borne pathogens (HIV, HTLV, HCV, HBV, CMV, HAV and Treponemapallidum) and fecal bacterial pathogens (Salmonella, Shigella, Yersinia,Campylobacter, E. coli 0157), ova and parasites, and other infectiousagents (Giardia, Cryptosporidium Cyclospora, Isospora) prior to stooldonation.

Donor stool was frozen shortly after donation and sampled for testing.At the time of use, approximately 75 g of donor stool was thawed andresuspended in 500 mL of non-bacteriostatic normal saline and mixed in asingle use glass or plastic blender. The resulting slurry wassequentially passed through sterile, disposable mesh screens that removeparticles of size 600, 300 and 200 microns. The slurry was thencentrifuged briefly (200 rcf for 4 min) to separate fibrous andparticulate materials, and the supernatant (containing bacterial cellsand spores) was transferred to a fresh container. Ethanol was added to afinal concentration of 50% and the resulting ˜1500 ml slurry wasincubated at room temperature for 1 hr with continuous mixing toinactivate vegetative bacterial cells. Midway through inactivation theslurry was transferred to a new bottle to ensure complete contact withthe ethanol. The solid matter was pelleted in a centrifuge and washed 3times with normal saline to remove residual ethanol. The final pelletwas resuspended in 100% sterile, USP glycerol at a minimum volume, andfilled into approximately 30 size 0 delayed release capsules(hypromellose DRcaps, Capsugel, Inc.) at 0.65 mL suspension each. Thecapsules were immediately capped and placed onto an aluminum freezingblock held at −80° C. via dry ice to freeze. The frozen capsules were inturn over-capsulated with size 00 DRcaps to enhance capsule stability,labeled, and placed into <−65° C. storage immediately. The final productwas stored at <−65° C. until the day and time of use. Encapsulatedproduct may be stored for indefinitely at <−65° C. On the day of dosingcapsules were warmed on wet ice for 1 to 2 hours to improvetolerability, and were then dosed with water ad libitium.

Patient 1 is a 45-year old woman with a history of C. difficileinfection and diarrhea for at least 1 year prior to treatment. She hasbeen previously treated with multiple courses of antibiotics followedeach time by recurrence of C. difficile-associated diarrhea.

Patient 2 is an 81-year old female who has experienced recurrent C.difficile infection for 6 months prior to treatment despite adequateantibiotic therapy following each recurrence.

24 hours prior to starting oral treatment, CDAD antibiotic therapy wasdiscontinued. Each patient received a colon preparation procedureintended to reduce the competing microbial burden in thegastrointestinal tract and to facilitate repopulation by the sporeforming organisms in the investigational product.

On the morning of the first treatment day, the patients received a doseof delayed release capsules containing the investigational product withwater ad libitum. Patients were requested to avoid food for 1 hourthereafter. The next day, the patient returned to the clinic to receivean additional dose. Patients were asked to avoid food for 4 hours priorto receiving their second dose and for 1 hour following dosing.

Both patients were followed closely for evidence of relapse or adversesymptoms following treatment. Patients were contacted by phone on Day 2,Day 4, and Weeks 1, 2 and 4 and each was queried about her generalstatus and the condition of her CDAD and related symptoms. Stool sampleswere collected at baseline and Weeks 1, 2, 4 and 8 post-treatment toassess changes in the gut microbiota via 16S sequencing and spore countwith methods explained previously (e.g. see Examples 11 and 12). Through4 weeks post treatment, each patient has gradually improved with noevidence of C. difficile recurrence.

Six other patients with recurrent C. difficile-associated diarrhea weretreated in a similar fashion, with no CDI recurrence and no requirementfor resumption of antibiotics (total of 8 patients). Additionally, therewere no treatment-related serious adverse events.

Example 19: Treatment of Fecal Suspensions with Ethanol or HeatDrastically Reduces Vegetative Cell Numbers and Results in an Enrichmentof Spore Forming Species

Treatment of a sample, preferably a human fecal sample, in a manner toinactivate or kill substantially all of the vegetative forms of bacteriapresent in the sample results in selection and enrichment of the sporefraction. Methods for inactivation can include heating, sonication,detergent lysis, enzymatic digestion (such as lysozyme and/or proteinaseK), ethanol or acid treatment, exposure to solvents (Tetrahydrofuran,1-butanol, 2-butanol, 1,2 propanediol, 1,3 propanediol, butanoate,propanoate, chloroform, dimethyl ether and a detergent like tritonX-100, diethyl ether), or a combination of these methods. To demonstratethe efficacy of ethanol induced inactivation of vegetative cells, a 10%fecal suspension was mixed with absolute ethanol in a 1:1 ratio andvortexed to mix for 1 min. The suspension was incubated at roomtemperature for 30 min, 1 h, 4 h or 24 h. After incubation thesuspension was centrifuged at 13,000 rpm for 5 min to pellet spores. Thesupernatant is discarded and the pellet is resuspended in equal volumeof PBS. Viable cells were measured as described below.

To demonstrate the efficacy of heat treatment on vegetative cellinactivation a 10-20% fecal suspension was incubated at 70 C, 80 C, 90 Cor 100 C for 10 min or 1 h.

After ethanol or heat treatment, remaining viable cells were measuredafter 24 h incubation on plates by determining the bacterial titer onBrucella blood agar (BBA) as a function of treatment and time (See FIG.6). Ethanol treatment for 1 h and 25 h have similar effects, reducingthe number of viable cells by approximately 4 logs, while increasingtemperature and time at high temperature leads to higher losses inviable cell number, with no colonies detectable at 100° C. at either 10min or 1 h. In this experiment no germinants were used. After severaldays of additional growth on plates, a number of colonies were pickedfrom these treated samples and identified by 16S rDNA analysis (e.g. seeExamples 11 and 12). These included known spore forming Clostridium spp.as well as species not previously reported to be spore formers includingRuminococcus bromii, and Anaerotruncus colihominis (Lawson, et al 2004),and a Eubacterium sp. (Table 4). See FIG. 6: Heat and ethanol treatmentsreduce cell viability

To demonstrate that vegetative cells are greatly reduced by ethanoltreatment, known non-spore forming bacteria are ethanol treated asdescribed previously (e.g. see Example 15) and viability was determinedby plating on BBA in anaerobic conditions (e.g. see Example 14). Fecalmaterial from four independent donors was exposed to 60 C for 5 min andsubsequently plated on three types of selective media under eitheraerobic (+O₂) or anaerobic conditions (−O₂) (BBA+aerobic, MacConkeylactose+aerobic, Bacteroides Bile esculin+anaerobic) to identify knownnonsporeforming Enterobacteria (survivors on MacConkey agar) andBacteroides fragilis group species (survivors on Bacteroides BileEsculin plates). The detectable limit for these assays was roughly 20cfu/mL. Germinants were not used in this experiment (FIG. 7). Bothethanol and heat inactivation greatly reduces the cell viability fromfecal material to the limit of detection under using MacConkey lactoseagar and BBE agar. The remaining cells identified on BBA media grown inanaerobic conditions comprise the non-germinant dependent spore formingspecies. See FIG. 7: Reduction in non-spore forming vegetative cells bytreatment at 60° C. for 5 min

Additionally, the ethanol treatment was shown to rapidly kill bothaerobic and non-spore forming anaerobic colony forming units in 10%fecal suspensions as determined by plating on rich (BBA) media. Thereduction of plated CFUs decreases four orders of magnitude in secondsas shown in FIG. 8.

See FIG. 8: Time Course Demonstrates Ethanol Reduces Both Anaerobic andAerobic Bacterial CFUs

Example 20: Species Identified and Isolated as Spore Formers by EthanolTreatment

To demonstrate that spore-forming species are enriched by heat orethanol treatment methods, a comparison of >7000 colony isolates wasperformed to identify species in a repeatable fashion (e.g., identifiedindependently in multiple preparations, see examples 1, 2, and 3) onlyisolated from fecal suspensions treated with 50% ethanol or heattreatment and not from untreated fecal suspensions (Table 5). These datademonstrate the ability to select for spore forming species from fecalmaterial, and identify organisms as spore formers not previouslydescribed as such in the literature. In each case, organisms were pickedas an isolated colony, grown anaerobically, and then subjected tofull-length 16S sequencing in order to assign species identity.

To further identify spore formers, ethanol treated fecal samples fromdonors A, B, C, D, E and F were plated to a variety of solid mediatypes, single colonies were picked and grown up in broth in a 96 wellformat (Table 6-11). The 16S rRNA gene was then amplified by PCR anddirect cycle sequencing was performed (See examples 11 and 12). The IDis based on the forward read from direct cycle sequencing of the 16SrRNA gene.

There is surprising heterogeneity in the microbiome from one individualto another (Clemente et al., 2012) and this has consequences fordetermining the potential efficacy of various donors to generate usefulspore compositions. The method described below is useful for screeningdonors when, for instance, a particular quantity or diversity of sporeforming organisms is useful or desired for repopulating the microbiomefollowing antibiotic treatment or treating a particular disease orcondition. Further, such screening is useful when there is a need toscreen donors for the purpose of isolating microorganisms capable ofspore formation, or when a purified preparation of spore formingorganisms is desired from a particular donor.

Total spore count is also a measure of potency of a particular donationor purified spore preparation and is vital to determine the quantity ofmaterial required to achieve a desired dose level. To understand thevariability in total spore counts, donor samples were collected andprocessed as described in prior examples. Donor spore counts in CFU/gwere then determined by growth on media plates at various titrations todetermine the spore content of the donation. Furthermore, DPA assayswere used to assess spore content (expressed as spore equivalents) asdescribed in Example 21. As seen in FIG. 9, there is as much as two logsdifference in an individual donor over time and can be up to three logsdifference between donors. One possible reason for the difference inspore content measures is that nonviable spores and non-germinablespores will not be observed by plating but will have measurable DPAcontent. Another possibility is the variability between species of DPAcontent in spores making some complex mixtures containing high DPAspores while other mixtures contain low DPA content spores. Selectingdonors with high spore counts is important in determining productivityof isolating spores from fecal donations by identifying preferreddonors.

See FIG. 9: Donation Spore Concentrations from Clinical Donors

A fresh fecal sample from donor F was treated as described in Example 15to generate an ethanol treated spore fraction, germinated withBHIS/Oxgall for 1 h as a described (e.g. see Example 13), then plated toa variety of media (e.g. See example 14). Colonies were picked with afocus on picking several of each type of morphologically distinct colonyon each plate to capture as much diversity as possible. Colonies arecounted on a plate of each media type with well isolated colonies suchthat the number of colony forming units per ml can be calculated (Table12). Colonies were picked into one of several liquid media and the 16SrDNA sequences (e.g. see Examples 11 and 12) were determined andanalyzed as described above. The number of unique OTUs for each mediatype is shown below with the media with the most unique OTUs at the top(Table 12). Combinations of 3 to 5 of the top 5 media types capturediversity, and some other can be chosen to target specific species ofinterest. Colony forming units can be calculated for a given speciesusing the 16S data, and could be used to determine whether a sufficientlevel of a given organism is present. The spore complement from Donor Fas determined in this experiment includes these 52 species as determinedby 16S sequencing (Table 12).

To screen human donors for the presence of a diversity of spore formingbacteria and/or for specific spore-forming bacteria, fecal samples wereprepared using germinants and selective plating conditions and colonieswere picked (e.g. see Examples 13 and 14) and analyzed for 16S diversityas described previously (see Examples 11 and 12). An assessment of donordiversity could include the cfu/ml of ethanol resistant cells on a givenmedia type, or cfu/ml of a given species using the 16S analysis ofcolonies picked from that media to determine the level of spores of agiven species of interest. This type of culture-based analysis could becomplemented by culture-independent methods such as qPCR with probesspecific to species or genera of interest or metagenomic sequencing ofspore preparations, or 16S profiling of spore preparations usingIllumina 16S variable region sequencing approaches (e.g. see Examples 11and 12).

Example 21: Quantification of Spore Concentrations Using DPA Assay

Methods to assess spore concentration in complex mixtures typicallyrequire the separation and selection of spores and subsequent growth ofindividual species to determine the colony forming units. The art doesnot teach how to quantitatively germinate all the spores in a complexmixture as there are many species for which appropriate germinants havenot been identified. Furthermore, sporulation is thought to be astochastic process as a result of evolutionary selection, meaning thatnot all spores from a single species germinate with same response togerminant concentration, time and other environmental conditions.Alternatively, a key metabolite of bacterial spores, dipicolinic acid(DPA) has been developed to quantify spores particles in a sample andavoid interference from fecal contaminants. The assay utilizes the factthat DPA chelates Terbium 3+ to form a luminescent complex (Fichtel etal, FEMS Microbiology Ecology, 2007; Kort et al, Applied andEnvironmental Microbiology, 2005; Shafaat and Ponce, Applied andEnvironmental Microbiology, 2006; Yang and Ponce, International Journalof Food Microbiology, 2009; Hindle and Hall, Analyst, 1999). Atime-resolved fluorescence assay detects terbium luminescence in thepresence of DPA giving a quantitative measurement of DPA concentrationin a solution.

To perform the assay 1 mL of the spore standard to be measured wastransferred to a 2 mL microcentrifuge tube. The samples were centrifugedat 13000 RCF for 10 min and the sample is washed in 1 mL steriledeionized H₂O. Wash an additional time by repeating the centrifugation.Transfer the 1 mL solution to hungate tubes and autoclave samples on asteam cycle for 30 min at 250 C. Add 100 uL of 30 uM TbCl₃ solution (400mM sodium acetate, pH 5.0, 30 μM TbCl₃) to the sample. Make serialdilutions of of the autoclaved material and measure the fluorescence ofeach sample by exciting with 275 nm light and measuring the emissionwavelength of 543 nm for an integration time of 1.25 ms and a 0.1 msdelay.

Purified spores are produced as described previously (e.g. seewww.epa.gov/pesticides/methods/MB-28-00.pdf). Serial dilutions ofpurified spores from C. bifermentans, C. sporogenes, and C. butyricumcultures were prepared and measured by plating on BBA media andincubating overnight at 37 C to determine CFU/ml. FIG. 10 shows thelinear correspondence across different spore producing bacteria acrossseveral logs demonstrating the DPA assay as means to assess sporecontent.

See FIG. 10: Linear Range of DPA Assay Compared to CFU Counts/Ml

The discrepancy for complex spore populations between spore countsmeasured by germinable spore CFU and by DPA has important implicationsfor determining the potency of an ethanol treated spore preparation forclinical use. Table AC shows spore content data from 3 different ethanoltreated spore preparations used to successfully treat 3 patientssuffering from recurrent C. difficile infection. The spore content ofeach spore preparation is characterized using the two described methods.

TABLE AC Spore quantitation for ethanol treated spore preparations usingspore CFU (SCFU) assay and DPA assay DPA SEq/30 Preparation SCFU/30capsules capsules Ratio SCFU/DPA Preparation 1 4.0 × 10{circumflex over( )}5 6.8 × 10{circumflex over ( )}7 5.9 × 10−3 Preparation 2 2.1 ×10{circumflex over ( )}7 9.2 × 10{circumflex over ( )}8 0.023Preparation 3 6.9 × 10{circumflex over ( )}9 9.6 × 10{circumflex over( )}9 0.72

What is immediately apparent is that spore content varies greatly per 30capsules. As measured by germinable SCFU, spore content varies bygreater than 10,000-fold. As measured by DPA, spore content varies bygreater than 100-fold. In the absence of the DPA assay, it would bedifficult to set a minimum dose for administration to a patient. Forinstance, without data from the DPA assay, one would conclude that aminimum effective dose of spores is 4×10⁵ or less using the SCFU assay(e.g. Preparation 1, Table AC). If that SCFU dose was used to normalizedosing in a clinical setting, however, then the actual spore doses givento patients would be much lower for other ethanol treated sporepreparations as measured as by the DPA assay (Table AD).

TABLE AD DPA doses in Table AC when normalized to 4 × 10⁵ SCFU per doseSCFU/ DPA SEq/30 Fraction of Preparation 1 Preparation 30 capsulescapsules Dose Preparation 1 4.0 × 10{circumflex over ( )}5 6.8 ×10{circumflex over ( )}7 1.0 Preparation 2 4.0 × 10{circumflex over( )}5 1.8 × 10{circumflex over ( )}7 0.26 Preparation 3 4.0 ×10{circumflex over ( )}5 5.6 × 10{circumflex over ( )}5 0.0082

It becomes clear from the variability of SCFU and DPA counts acrossvarious donations that using SCFU as the measure of potency would leadto significant underdosing in certain cases. For instance, setting adose specification of 4×10⁵ SCFU (the apparent effective dose from donorPreparation 1) for product Preparation 3 would lead to a potentialunderdosing of more than 100-fold. This can be rectified only by settingpotency specifications based on the DPA assay, which better reflectstotal spore counts in an ethanol treated spore preparation. Theunexpected finding of this work is that the DPA assay is uniquely suitedto set potency and determine dosing for an ethanol treated sporepreparation.

Example 22: Demonstration of Enhanced Growth with a Germinant

To enhance the ethanol treated spores germination capability anddemonstrate spore viability, spores from three different donors weregerminated by various treatments and plated on various media.Germination with BHIS/oxgall (BHIS ox), Ca-DPA, rumen fluid (RF),simulated gastric fluid (SGF), mucin medium (Muc), fetal bovine serum(FBS), or thioglycollate (Thi) for 1 hour at 37 C in anaerobicconditions was performed as described previously (e.g. see Examples 13and 14) with samples derived from two independent donors (FIG. 11). Thespore-germinant mixture was serially diluted and plated on differentplate media including BBA, Sweet B, Sweet B+lysozyme (2 ug/ml), M2GSCand M2GSC+lysozyme (2 ug/ml) as previously described (e.g. see Examples13 and 14) to determine spore germination. Colony forming units weretallied and titers were determined using standard techniques by oneskilled in the art. As FIG. 11 shows, maximum colony forming units arederived from BHI-oxgall treatment. This germination treatment alsogreatly increases the diversity as measured by the number of OTUsidentified when samples were submitted for 16S sequencing (e.g. seeExamples 11 and 12) compared to plating without a germination step (FIG.12). As shown in FIG. 11: Different germinant treatments have variableeffects on CFU counts from donor A (upper left) and donor B (lowerright). The Y-Axes are spore CFU per ml. As shown in FIG. 12: Germinatesgreatly increase the diversity of cultured spore forming OTUs.

To test the effect of heat activation to promote germination, ethanoltreated fecal samples were treated for 15 min at room temperature, 55 C,65 C, 75 C or 85 C from three different donors and germinatedsubsequently with BHIS+Oxgall for 1 hr at 37 C then plated on BBA media(FIG. 13) as previously described (e.g. see Examples 13 and 14).Pretreatment at room temperature produced equal if not more spores thanthe elevated temperatures in all three donors. The temperature ofgerminating was also examined by incubating samples at room temperatureor 37 C for 1 hr in anaerobic conditions before plating on BBA. Nodifference in the number of CFUs was observed between the twoconditions. Lysozyme addition to the plates (2 ug/ml) was also tested ona single donor sample by the testing of various activation temperaturefollowed by an incubation in the presence or absence of lysozyme. Theaddition of lysozyme had a small effect when plated on Sweet B or M2GSCmedia but less so than treatment with BHIS oxgall without lysozyme for 1hr (FIG. 14).

As shown in FIG. 13: Heat Activation as a germination treatment withBHIS+oxgall. As shown in FIG. 14: Effect of lysozyme slightly enhancesgermination.

Germination time was also tested by treating a 10% suspension of asingle donor ethanol treated feces (e.g. see Example 15) incubated ineither BHIS, taurocholate, oxgall, or germix for 0, 15, 30, or 60minutes and subsequently plated on BHIS, EYA, or BBA media (e.g. seeExamples 13 and 14). 60 minutes resulted in the most CFU units acrossall various combinations germinates and plate media tested.

Example 23: Demonstrating Efficacy of Germinable and SporulatableFractions of Ethanol Treated Spores

To define methods for characterization and purification, and to improve(e.g., such as by modulating the diversity of the compositions) theactive spore forming ecology derived from fecal donations, the ethanoltreated spore population (as described in Example 15) was furtherfractionated. A “germinable fraction” was derived by treating theethanol-treated spore preparation with oxgall, plating to various solidmedia, and then, after 2 days or 7 days of growth, scraping all thebacterial growth from the plates into 5 mL of PBS per plate to generatea bacterial suspension. A “sporulatable fraction” was derived as aboveexcept that the cells were allowed to grow on solid media for 2 days or7 days (the time was extended to allow sporulation, as is typical insporulation protocols), and the resulting bacterial suspension wastreated with 50% ethanol to derive a population of “sporulatable”spores, or species that were capable of forming spores. In preparingthese fractions, fecal material from donor A was used to generate anethanol treated spore preparation as previously described in Example 15;then spore content was determined by DPA assay and CFU/ml grown onvarious media (FIG. 15) as previously described (see Example 21). SeeFIG. 15: Spores initially present in ethanol treated spore preparationas measured by DPA and CFU/ml grown on specified media.

To characterize the fraction that is sporulatable, the 2 day and 7 day“germinable” fractions were assessed for CFU and DPA content before andafter ethanol treatment to generate a spore fraction. Bacterialsuspensions were treated with ethanol, germinated with Oxgall, andplated on the same types of media that the “germinable” fraction wasgrown on. DPA data showed that growth on plates for 2 and 7 daysproduced the same amount of total spores. Colonies on the several typesof media were picked for 16S sequence analysis to identify the sporeforming bacteria present (Table 13).

A 2 day “germinable” fraction and a 7 day “sporulatable” fraction wereused as a treatment in the mouse prophylaxis assay as described (e.g.see Example 16). As a control, a 10% fecal suspension prepared from adonor (Donor B) was also administered to mice to model fecal microbiotatransplant (FMT). Weight loss and mortality of the various test andcontrol arms of the study are plotted in FIG. 17 and summarized in Table15 which also contains the dosing information. The data clearly showsboth the “germinable” and “sporulatable” fractions are efficacious inproviding protection against C. difficile challenge in a prophylaxismouse model (e.g. see Example 16). The efficacy of these fractionsfurther demonstrates that the species present are responsible for theefficacy of the spore fraction, as the fractionation further dilutes anypotential contaminant from the original spore preparation.

See FIG. 16: Titer of “germinable” fraction after 2 days (left) andSporulatable fraction (right) by DPA and CFU/ml. The “sporulatable”fraction made following 7 days of growth was measured as previouslydescribed using germination and growth assays or DPA content aspreviously described (see Example 21).

The species present in the “germinable” and “sporulatable” fractionswere determined by full length 16S sequencing of colony picks and by 16SNGS sequencing of the fractions themselves. The colony pick dataindicate Clostridium species are very abundant in both fractions, whilethe NGS data reveal other spore forming organisms that are typicallyfound in ethanol treated spore preparations are present.

Results are shown in the following: See Table 13. Species identified as“germinable” and “sporulatable” by colony picking approach. See TableYYY. Species identified as “germinable” using 16S-V4 NGS approach. SeeTable ZZZ. Species identified as “sporulatable” using 16s-V4 NGSapproach. See FIG. 17: Mouse prophylaxis model demonstrates “germinable”and “sporulatable” preparations are protective against C. difficilechallenge. Each plot tracks the change in the individual mouse's weightrelative to day −1 over the course of the experiment. The number ofdeaths over the course of the experiment is indicated at the top of thechart and demonstrated by a line termination prior to day 6. The toppanels (from left to right) are the vehicle control arm, the fecalsuspension arm, and the untreated naive control arm, while the bottompanels are the ethanol treated, gradient purified spore preparation; theethanol treated, gradient purified, “germinable” preparation, andethanol treated, gradient purified, “sporulatable” preparation. SeeTable 15: Results of the prophylaxis mouse model and dosing information

Example 24: Donor Pooling Efficacy in Prophylaxis Mouse

To test the efficacy and dosing of pooled donor samples the C. difficileprophylaxis mouse model (e.g., see Example 16) is used with donationsmixed from two or more donor samples as previously described. Weightloss and mortality with the mixed spore product versus the spore productderived from a single donor at the various dosing is determine whetherthe two treatment schemes are equivalent or one is significantly betterthan the other.

Dosing of the spore product derived from a single or multiple donors isbetween 1E4 to 1E10 CFU/ml. The spore product is mixed from productderived from any number of donors ranging from 1-10 at either equalconcentrations or different known concentrations.

Additionally, this method can be used to expand the spore fraction forproduction purposes. For production purposes, an enriched spore fraction(e.g. —a purified and EtOH treated fraction of a fecal sample) ispreserved in multiple aliquots to form a bank of viable spore-formingorganisms. An aliquot of this bank is then recovered by germinanttreatment followed by cultivation in a medium, and under conditions,that are broadly permissive for spore-forming organisms and encouragesporulation. After a suitable amplification time, the amplified bacteriaincluding spores are harvested, and this preparation is solvent orheat-treated to isolate the spore fraction. This fraction may be furtherpurified away from non-spore forms and culture constituents. The processof amplification, spore isolation and optional purification may berepeated at increasing scales to generate large quantities for furtheruse. When enough germinable/sporulatable material has been accumulatedby amplification, it may be further purified, concentrated or diluted,and/or preserved to a state suitable for further use, e.g.—clinicaldosing.

Features may be incorporated into the above process to make it suitablefor further utility, especially for product applications such asclinical use. The production of the initial spore fraction may beconducted under controlled conditions (cGMP's) and validated to removenon-spore organisms to a high degree. The germination may be conductedusing reagents that are more standardizable than natural products suchas oxgall, e.g.—synthetic mixtures of bile salts. Amplification may bedone using media with components that are preferred for clinical safety,e.g.—sourced from qualified animals, or non-animal sourced. Conditionsmay be arranged so as to ensure consistent compositions of sporulatedorganisms, are less prone to contamination, and are more amenable toscale-up, e.g.—closed stirred fermenters with feedback control loops.Sporulated organisms from the process may be isolated using proceduresthat alone or combined stringently eliminate non-spores and otherprocess residuals, e.g.—solvent treatment, aqueous two-phase extraction,and/or 60° C. long-time heat treatment. Preservation may involveaddition of excipients and/or adjustment of conditions to enableconversion to a preferred dosage form amenable to long-term shelfstorage, e.g.—addition of trehalose, followed by lyophilization or spraydrying, further blending of the powder with microcrystalline cellulose,and encapsulation in a gelatin capsule to form an orally dosableproduct.

Example 25: Engraftment, Augmentation and Reduction of Pathogen Carriagein Patients Treated with Spore Compositions

Complementary genomic and microbiological methods were used tocharacterize the composition of the microbiota from Patient 1, 2, 3, 4,and 5, 6, 7, 8, 9, and 10 at pretreatment (pretreatment) and on up to 4weeks post-treatment.

To determine the OTUs that engraft from treatment with an ethanoltreated spore preparation in the patients and how their microbiomechanged in response, the microbiome was characterized by 16S-V4sequencing prior to treatment (pretreatment) with an ethanol treatedspore preparation and up to 25 days after receiving treatment. Asexample, the treatment of patient 1 with an ethanol treated sporepreparation led to the engraftment of OTUs from the spore treatment andaugmentation in the microbiome of the patient (FIG. 18 and FIG. 19). Byday 25 following treatment, the total microbial carriage was dominatedby species of the following taxonomic groups: Bacteroides, Sutterella,Ruminococcus, Blautia, Eubacterium, Gemmiger/Faecalibacterium, and thenon-sporeforming Lactobacillus (see Table 16 and Table 2 for specificOTUs). The first two genera represent OTUs that do not form spores whilethe latter taxonomic groups represent OTUs that are believed to formspores.

Patient treatment with the ethanol treated spore preparation leads tothe establishment of a microbial ecology that has greater diversity thanprior to treatment (FIG. 18). Genomic-based microbiome characterizationconfirmed engraftment of a range of OTUs that were absent in the patientpretreatment (Table 16). These OTUs comprised both bacterial speciesthat were capable and not capable of forming spores, and OTUs thatrepresent multiple phylogenetic clades. Organisms absent in Patient 1pre-treatment either engraft directly from the ethanol treated sporefraction or are augmented by the creation of a gut environment favoringa healthy, diverse microbiota. Furthermore, Bacteroides fragilis groupspecies were increased by 4 and 6 logs in patients 1 and 2 (FIG. 20).

OTUs that comprise an augmented ecology are not present in the patientprior to treatment and/or exist at extremely low frequencies such thatthey do not comprise a significant fraction of the total microbialcarriage and are not detectable by genomic and/or microbiological assaymethods. OTUs that are members of the engrafting and augmented ecologieswere identified by characterizing the OTUs that increase in theirrelative abundance post treatment and that respectively are: (i) presentin the ethanol treated spore preparation and absent in the patientpretreatment, or (ii) absent in the ethanol treated spore preparation,but increase in their relative abundance through time post treatmentwith the preparation due to the formation of favorable growth conditionsby the treatment. Notably, the latter OTUs can grow from low frequencyreservoirs in the patient, or be introduced from exogenous sources suchas diet. OTUs that comprise a “core” augmented or engrafted ecology canbe defined by the percentage of total patients in which they areobserved to engraft and/or augment; the greater this percentage the morelikely they are to be part of a core ecology responsible for catalyzinga shift away from a dysbiotic ecology. The dominant OTUs in an ecologycan be identified using several methods including but not limited todefining the OTUs that have the greatest relative abundance in eitherthe augmented or engrafted ecologies and defining a total relativeabundance threshold. As example, the dominant OTUs in the augmentedecology of Patient-1 were identified by defining the OTUs with thegreatest relative abundance, which together comprise 60% of themicrobial carriage in this patient's augmented ecology.

See FIG. 18: Microbial diversity measured in the ethanol treated sporetreatment sample and patient pre- and post-treatment samples. Totalmicrobial diversity is defined using the Chao1 Alpha-Diversity Index andis measured at different genomic sampling depths to confirm adequatesequence coverage to assay the microbiome in the target samples. Thepatient pretreatment (purple) harbored a microbiome that wassignificantly reduced in total diversity as compared to the ethanoltreated spore treatment (red) and patient post treatment at days 5(blue), 14 (orange), and 25 (green).

See FIG. 19: Patient microbial ecology is shifted by treatment with anethanol treated spore treatment from a dysbiotic state to a state ofhealth. Principle Coordinates Analysis based on the total diversity andstructure of the microbiome (Bray-Curtis Beta-Diversity) of the patientpre- and post-treatment delineates that the engraftment of OTUs from thespore treatment and the augmentation of the patient microbial ecologyleads to a microbial ecology that is distinct from both the pretreatmentmicrobiome and the ecology of the ethanol treated spore treatment (Table16).

See FIG. 20: Augmentation of Bacteroides species in patients. Comparingthe number of Bacteroides fragilis groups species per cfu/g of fecespre-treatment and in week 4 post treatment reveals an increase of 4 logsor greater. The ability of 16S-V4 OTU identification to assign an OTU asa specific species depends in part on the resolution of the 16S-V4region of the 16S gene for a particular species or group of species.Both the density of available reference 16S sequences for differentregions of the tree as well as the inherent variability in the 16S genebetween different species will determine the definitiveness of ataxonomic annotation to a given sequence read. Given the topologicalnature of a phylogenetic tree and that the tree represents hierarchicalrelationships of OTUs to one another based on their sequence similarityand an underlying evolutionary model, taxonomic annotations of a readcan be rolled up to a higher level using a clade-based assignmentprocedure (Table 1). Using this approach, clades are defined based onthe topology of a phylogenetic tree that is constructed from full-length16S sequences using maximum likelihood or other phylogenetic modelsfamiliar to individuals with ordinary skill in the art of phylogenetics.Clades are constructed to ensure that all OTUs in a given clade are: (i)within a specified number of bootstrap supported nodes from one another(generally, 1-5 bootstraps), and (ii) within a 5% genetic similarity.OTUs that are within the same clade can be distinguished as geneticallyand phylogenetically distinct from OTUs in a different clade based on16S-V4 sequence data. OTUs falling within the same clade areevolutionarily closely related and may or may not be distinguishablefrom one another using 16S-V4 sequence data. The power of clade basedanalysis is that members of the same clade, due to their evolutionaryrelatedness, play similar functional roles in a microbial ecology suchas that found in the human gut. Compositions substituting one specieswith another from the same clade are likely to have conserved ecologicalfunction and therefore are useful in the present invention.

Stool samples were aliquoted and resuspended 10× vol/wt in either 100%ethanol (for genomic characterization) or PBS containing 15% glycerol(for isolation of microbes) and then stored at −80° C. until needed foruse. For genomic 16S sequence analysis colonies picked from plateisolates had their full-length 16S sequence characterized as describedin Examples 11 and 12, and primary stool samples were prepared targetingthe 16S-V4 region using the method for heterogeneous samples in Example10.

Notably, 16S sequences of isolates of a given OTU are phylogeneticallyplaced within their respective clades despite that the actual taxonomicassignment of species and genus may suggest they are taxonomicallydistinct from other members of the clades in which they fall.Discrepancies between taxonomic names given to an OTU is based onmicrobiological characteristics versus genetic sequencing are known toexist from the literature. The OTUs footnoted in this table are known tobe discrepant between the different methods for assigning a taxonomicname.

Engraftment of OTUs from the ethanol treated spore preparation treatmentinto the patient as well as the resulting augmentation of the residentmicrobiome led to a significant decrease in and elimination of thecarriage of pathogenic species other than C. difficile in the patient.16S-V4 sequencing of primary stool samples demonstrated that atpretreatment, 20% of reads were from the genus Klebsiella and anadditional 19% were assigned to the genus Fusobacterium. These strikingdata are evidence of a profoundly dysbiotic microbiota associated withrecurrent C. difficile infection and chronic antibiotic use. In healthyindividuals, Klebsiella is a resident of the human microbiome in onlyabout 2% of subjects based on an analysis of HMP database(www.hmpdacc.org), and the mean relative abundance of Klebsiella is onlyabout 0.09% in the stool of these people. It's surprising presence at20% relative abundance in Patient 1 before treatment is an indicator ofa proinflammatory gut environment enabling a “pathobiont” to overgrowand outcompete the commensal organisms normally found in the gut.Similarly, the dramatic overgrowth of Fusobacterium indicates aprofoundly dysbiotic gut microbiota. One species of Fusobacterium, F.nucleatum (an OTU phylogenetically indistinguishable from Fusobacteriumsp. 3_1_33 based on 16S-V4), has been termed “an emerging gut pathogen”based on its association with IBD, Crohn's disease, and colorectalcancer in humans and its demonstrated causative role in the developmentof colorectal cancer in animal models [Allen-Vercoe, Gut Microbes (2011)2:294-8]. Importantly, neither Klebsiella nor Fusobacterium was detectedin the 16S-V4 reads by Day 25 (Table 18).

To further characterize the colonization of the gut by Klebsiella andother Enterobacteriaceae and to speciate these organisms, pretreatmentand Day 25 fecal samples stored at −80 C as PBS-glycerol suspensionswere plated on a variety of selective media including MacConkey lactosemedia (selective for gram negative enterobacteria) and Simmons CitrateInositol media (selective for Klebsiella spp) [Van Cregten et al, J.Clin. Microbiol. (1984) 20: 936-41]. Enterobacteria identified in thepatient samples included K. pneumoniae, Klebsiella sp. Co_9935 and E.coli. Strikingly, each Klebsiella species was reduced by 2-4 logswhereas E. coli, a normal commensal organism present in a healthymicrobiota, was reduced by less than 1 log (Table 19). This decrease inKlebsiella spp. carriage is consistent across multiple patients (Table19). Four separate patients were evaluated for the presence ofKlebsiella spp. pre treatment and 4 weeks post treatment. Klebsiellaspp. were detected by growth on selective Simmons Citrate Inositol mediaas previously described. Serial dilution and plating, followed bydetermining cfu/mL titers of morphologically distinct species and 16Sfull length sequence identification of representatives of those distinctmorphological classes, allowed calculation of titers of specificspecies.

The genus Bacteroides is an important member of the gastrointestinalmicrobiota; 100% of stool samples from the Human Microbiome Projectcontain at least one species of Bacteroides with total relativeabundance in these samples ranging from 0.96% to 93.92% with a medianrelative abundance of 52.67% (www.hmpdacc.org reference data setHMSMCP). Bacteroides in the gut has been associated with amino acidfermentation and degradation of complex polysaccharides. Its presence inthe gut is enhanced by diets rich in animal-derived products as found inthe typical western diet [David, L. A. et al, Nature (2013)doi:10.1038/nature12820]. Strikingly, prior to treatment, fewer than0.008% of the 16S-V4 reads from Patient 1 mapped to the genusBacteroides strongly suggesting that Bacteroides species were absent orthat viable Bacteroides were reduced to an extremely minor component ofthe patient's gut microbiome. Post treatment, ≧42% of the 16S-V4 readscould be assigned to the genus Bacteroides within 5 days of treatmentand by Day 25 post treatment 59.48% of the patients gut microbiome wascomprised of Bacteroides. These results were confirmed microbiologicallyby the absence of detectable Bacteroides in the pretreatment sampleplated on two different Bacteroides selective media: Bacteroides BileEsculin (BBE) agar which is selective for Bacteroides fragilis groupspecies [Livingston, S. J. et al J. Clin. Microbiol (1978). 7: 448-453]and Polyamine Free Arabinose (PFA) agar [Noack et al. J. Nutr. (1998)128: 1385-1391; modified by replacing glucose with arabinose]. Thehighly selective BBE agar had a limit of detection of <2×10³ cfu/g,while the limit of detection for Bacteroides on PFA agar wasapproximately 2×10⁷ cfu/g due to the growth of multiple non-Bacteroidesspecies in the pretreatment sample on that medium. Colony counts ofBacteroides species on Day 25 were up to 2×10¹⁰ cfu/g, consistent withthe 16S-V4 sequencing, demonstrating a profound reconstitution of thegut microbiota in Patient 1 (Table 20).

The significant abundance of Bacteroides in Patient 1 on Day 25 (and asearly as Day 5 as shown by 16S-V4 sequencing) is remarkable. ViableBacteroides fragilis group species were not present in the ethanoltreated spore population based on microbiological plating (limit ofdetection of 10 cfu/ml). Thus, administration of the ethanol treatedspore population to Patient 1 resulted not only in the engraftment ofspore-forming species, but also the restoration of high levels ofnon-spore forming species commonly found in healthy individuals throughthe creation of a niche that allowed for the repopulation of Bacteroidesspecies. These organisms were most likely either present at extremelylow abundance in the GI tract of Patient 1, or present in a reservoir inthe GI tract from which they could rebound to high titer. Those speciesmay also be reinoculated via oral uptake from food following treatment.We term this healthy repopulation of the gut with OTUs that are notpresent in the ethanol treated spore population “Augmentation.”Augmentation is an important phenomenon in that it shows the ability touse an ethanol treated spore ecology to restore a healthy microbiota byseeding a diverse array or commensal organisms beyond the actualcomponent organisms in the ethanol treated spore population itself;specifically the spore treatment itself and the engraftment of OTUs fromthe spore composition create a niche that enables the outgrowth of OTUsrequired to shift a dysbiotic microbiome to a microbial ecology that isassociated with health. The diversity of Bacteroides species and theirapproximate relative abundance in the gut of Patient 1 is shown in Table21, comprising at least 8 different species.

See FIG. 21: Species Engrafting versus Species Augmenting in patientsmicrobiomes after treatment with an ethanol-treated spore population.Relative abundance of species that engrafted or augmented as describedwere determined based on the number of 16S sequence reads. Each plot isfrom a different patient treated with the ethanol-treated sporepopulation for recurrent C. difficile.

The impact of ethanol treated spore population treatment on carriage ofimipenem resistant Enterobacteriaceae was assessed by platingpretreatment and Day 28 clinical samples from Patients 2, 4 and 5 onMacConkey lactose plus 1 ug/mL of imipenem. Resistant organisms werescored by morphology, enumerated and DNA was submitted for full length16S rDNA sequencing as described above. Isolates were identified asMorganella morganii, Providencia rettgeri and Proteus pennerii. Each ofthese are gut commensal organisms; overgrowth can lead to bacteremiaand/or urinary tract infections requiring aggressive antibiotictreatment and, in some cases, hospitalization [Kim, B-N, et al Scan J.Inf Dis (2003) 35: 98-103; Lee, I-K and Liu, J-W J. Microbiol ImmunolInfect (2006) 39: 328-334; O'Hara et al, Clin Microbiol Rev (2000) 13:534]. The titer of organisms at pretreatment and Day 28 by patient isshown in Table 22. Importantly, administration of the ethanol treatedspore preparation resulted in greater than 100-fold reduction in 4 of 5cases of Enterobacteriaceae carriage with multiple imipenem resistantorganisms (Table 22).

In addition to speculation and enumeration, multiple isolates of eachorganism from Patient 4 were grown overnight in 96-well trays containinga 2-fold dilution series of imipenem in order to quantitativelydetermine the minimum inhibitory concentration (MIC) of antibiotic.Growth of organisms was detected by light scattering at 600 nm on aSpectraMax M5e plate reader. In the clinical setting, these species areconsidered resistant to imipenem if they have an MIC of 1 ug/mL orgreater. M. morganii isolates from pretreatment samples from Patient Dhad MICs of 2-4 ug/mL and P. pennerii isolates had MICs of 4-8 ug/mL.Thus the ethanol treated spore population administered to Patient 4caused the clearance of 2 imipenem resistant organisms (Table 16).

Example 26. Enrichment and Purification of Bacteria

To purify individual bacterial strains, dilution plates were selected inwhich the density enables distinct separation of single colonies.Colonies were picked with a sterile implement (either a sterile loop ortoothpick) and re-streaked to BBA or other solid media. Plates wereincubated at 37° C. for 3-7 days. One or more well-isolated singlecolonies of the major morphology type were re-streaked. This process wasrepeated at least three times until a single, stable colony morphologyis observed. The isolated microbe was then cultured anaerobically inliquid media for 24 hours or longer to obtain a pure culture of 10⁶-10¹⁰cfu/ml. Liquid growth medium might include Brain Heart Infusion-basedmedium (Atlas, Handbook of Microbiological Media, 4th ed, ASM Press,2010) supplemented with yeast extract, hemin, cysteine, andcarbohydrates (for example, maltose, cellobiose, soluble starch) orother media described previously (e.g. see example 14). The culture wascentrifuged at 10,000×g for 5 min to pellet the bacteria, the spentculture media was removed, and the bacteria were resuspended in sterilePBS. Sterile 75% glycerol was added to a final concentration of 20%. Analiquot of glycerol stock was titered by serial dilution and plating.The remainder of the stock was frozen on dry ice for 10-15 min and thenplaced at −80 C for long term storage.

Example 27. Cell Bank Preparation

Cell banks (RCBs) of bacterial strains were prepared as follows.Bacterial strains were struck from −80° C. frozen glycerol stocks toBrucella blood agar with Hemin or Vitamin K (Atlas, Handbook ofMicrobiological Media, 4th ed, ASM Press, 2010), M2GSC (Atlas, Handbookof Microbiological Media, 4th ed, ASM Press, 2010) or other solid growthmedia and incubated for 24 to 48 h at 37° C. in an anaerobic chamberwith a gas mixture of H₂: CO₂: N₂ of 10:10:80. Single colonies were thenpicked and used to inoculate 250 ml to 1 L of Wilkins-Chalgren broth,Brain-Heart Infusion broth, M2GSC broth or other growth media, and grownto mid to late exponential phase or into the stationary phase of growth.Alternatively, the single colonies may be used to inoculate a pilotculture of 10 ml, which were then used to inoculate a large volumeculture. The growth media and the growth phase at harvest were selectedto enhance cell titer, sporulation (if desired) and phenotypes thatmight be associated desired in vitro or in vivo. Optionally, Cultureswere grown static or shaking, depending which yielded maximal celltiter. The cultures were then concentrated 10 fold or more bycentrifugation at 5000 rpm for 20 min, and resuspended in sterilephosphate buffered saline (PBS) plus 15% glycerol. 1 ml aliquots weretransferred into 1.8 ml cryovials which were then frozen on dry ice andstored at −80 C. The identity of a given cell bank was confirmed by PCRamplification of the 16S rDNA gene, followed by Sanger direct cyclesequencing, and comparison to a curated rDNA database to determine ataxonomic ID. Each bank was confirmed to yield colonies of a singlemorphology upon streaking to Brucella blood agar or M2GSC agar. Whenmore than one morphology was observed, colonies were confirmed to be theexpected species by PCR and sequencing analysis of the 16S rDNA gene.Variant colony morphologies can be observed within pure cultures, and ina variety of bacteria the mechanisms of varying colony morphologies havebeen well described (van der Woude, Clinical Microbiology Reviews,17:518, 2004), including in Clostridium species (Wadsworth-KTL AnaerobicBacteriology Manual, 6th Ed, Jousimie-Somer, et al 2002). For obligateanaerobes, RCBs were confirmed to lack aerobic colony forming units at alimit of detection of 10 cfu/ml.

Example 28. Titer Determination

The number of viable cells per ml was determined on the freshlyharvested, washed and concentrated culture by plating serial dilutionsof the RCB to Brucella blood agar or other solid media, and varied from10⁶ to 10¹⁰ cfu/ml. The impact of freezing on viability was determinedby titering the banks after one or two freeze-thaw cycles on dry ice orat −80° C., followed by thawing in an anaerobic chamber at roomtemperature. Some strains displayed a 1-3 log drop in viable cfu/mlafter the 1st and/or 2nd freeze thaw, while the viability of others wereunaffected.

Example 29. Preparation of Bacterial Compositions

Individual strains were typically thawed on ice and combined in ananaerobic chamber to create mixtures, followed by a second freeze at−80° C. to preserve the mixed samples. When making combinations ofstrains for in vitro or in vivo assays, the cfu in the final mixture wasestimated based on the second freeze-thaw titer of the individualstrains. For experiments in rodents, strains may be combined at equalcounts in order to deliver between 1e4 and 1e10 per strain.Additionally, some bacteria may not grow to sufficient titer to yieldcell banks that allowed the production of compositions where allbacteria were present at 1e10.

Example 30. Identification of Keystone OTUs and Functions

The human body is an ecosystem in which the microbiota, and themicrobiome, play a significant role in the basic healthy function ofhuman systems (e.g. metabolic, immunological, and neurological). Themicrobiota and resulting microbiome comprise an ecology ofmicroorganisms that co-exist within single subjects interacting with oneanother and their host (i.e., the mammalian subject) to form a dynamicunit with inherent biodiversity and functional characteristics. Withinthese networks of interacting microbes (i.e. ecologies), particularmembers can contribute more significantly than others; as such thesemembers are also found in many different ecologies, and the loss ofthese microbes from the ecology can have a significant impact on thefunctional capabilities of the specific ecology. Robert Paine coined theconcept “Keystone Species” in 1969 (see Paine RT. 1969. A note ontrophic complexity and community stability. The American Naturalist 103:91-93.) to describe the existence of such lynchpin species that areintegral to a given ecosystem regardless of their abundance in theecological community. Paine originally describe the role of the starfishPisaster ochraceus in marine systems and since the concept has beenexperimentally validated in numerous ecosystems.

Keystone OTUs and/or Functions are computationally-derived by analysisof network ecologies elucidated from a defined set of samples that sharea specific phenotype. Keystone OTUs and/or Functions are defined as allNodes within a defined set of networks that meet two or more of thefollowing criteria. Using Criterion 1, the node is frequently observedin networks, and the networks in which the node is observed are found ina large number of individual subjects; the frequency of occurrence ofthese Nodes in networks and the pervasiveness of the networks inindividuals indicates these Nodes perform an important biologicalfunction in many individuals. Using Criterion 2, the node is frequentlyobserved in networks, and each the networks in which the node isobserved contain a large number of Nodes—these Nodes are thus“super-connectors”, meaning that they form a nucleus of a majority ofnetworks and as such have high biological significance with respect totheir functional contributions to a given ecology. Using Criterion 3,the node is found in networks containing a large number of Nodes (i.e.they are large networks), and the networks in which the node is foundoccur in a large number of subjects; these networks are potentially ofhigh interest as it is unlikely that large networks occurring in manyindividuals would occur by chance alone strongly suggesting biologicalrelevance. Optionally, the required thresholds for the frequency atwhich a node is observed in network ecologies, the frequency at which agiven network is observed across subject samples, and the size of agiven network to be considered a Keystone node are defined by the 50th,70th, 80th, or 90th percentiles of the distribution of these variables.Optionally, the required thresholds are defined by the value for a givenvariable that is significantly different from the mean or median valuefor a given variable using standard parametric or non-parametricmeasures of statistical significance. In another embodiment a Keystonenode is defined as one that occurs in a sample phenotype of interestsuch as but not limited to “health” and simultaneously does not occur ina sample phenotype that is not of interest such as but not limited to“disease.” Optionally, a Keystone Node is defined as one that is shownto be significantly different from what is observed using permuted testdatasets to measure significance.

Example 31. Identifying the Core Ecology from the Ethanol Treated SporePreparation

Ten different ethanol treated spore preparations were made from 6different donors (as described in Example 15). The spore preparationswere used to treat 10 patients, each suffering from recurrent C.difficile infection. Patients were identified using theinclusion/exclusion criteria described in Example 18, and donors wereidentified using the criteria described in Example 1. None of thepatients experienced a relapse of C. difficile in the 4 weeks of followup after treatment, whereas the literature would predict that 70-80% ofsubjects would experience a relapse following cessation of antibiotic[Van Nood, et al, NEJM (2013)]. Thus, the ethanol treated sporepreparations derived from multiple different donors and donations showedremarkable clinical efficacy.

To define the Core Ecology underlying the remarkable clinical efficacyof the ethanol treated spore preparation, the following analysis wascarried out. The OTU composition of the spore preparation was determinedby 16S-V4 rDNA sequencing and computational assignment of OTUs perExample 12. A requirement to detect at least ten sequence reads in theethanol treated spore preparation was set as a conservative threshold todefine only OTUs that were highly unlikely to arise from errors duringamplification or sequencing. Methods routinely employed by thosefamiliar to the art of genomic-based microbiome characterization use aread relative abundance threshold of 0.005% (see e.g. Bokulich, A. etal. 2013. Quality-filtering vastly improves diversity estimates fromIllumina amplicon sequencing. Nature Methods 10: 57-59), which wouldequate to ≦2 reads given the sequencing depth obtained for the samplesanalyzed in this example, as cut-off which is substantially lower thanthe ≦10 reads used in this analysis. All taxonomic and clade assignmentswere made for each OTU as described in Examples 12. The resulting listof OTUs, clade assignments, and frequency of detection in the sporepreparations are shown in Table GB. OTUs that engraft in a treatedpatients and the percentage of patients in which they engraft aredenoted, as are the clades, spore forming status, and Keystone OTUstatus. Starred OTUs occur in ≦80% of the ethanol preps and engraft in≦50% of the treated patients.

TABLE GB OTUs detected by a minimum of ten 16S-V4 sequence reads in atleast a one ethanol treated spore preparation (pan- microbiome). % of %of Spore Patients Preps with OTU Spore Keystone OTU Clade OTU EngraftsFormer OTU Prevotella_maculosa clade_104 10% 0% N N Prevotella_copriclade_168 20% 0% N N Bacteroides_caccae clade_170 30% 0% N YBifidobacterium_sp_TM_7* clade_172 90% 60% N N Bifidobacterium_gallicumclade_172 70% 20% N N Bifidobacterium_dentium clade_172 50% 0% N NLactobacillus_casei clade_198 20% 10% N N Actinomyces_odontolyticusclade_212 20% 30% N N Clostridium_colicanis clade_223 10% 10% Y NClostridiales_sp_SS3_4* clade_246 100% 70% Y N Clostridium_sporogenesclade_252 40% 40% Y N Clostridium_butyricum clade_252 20% 20% Y NClostridium_disporicum clade_253 40% 30% Y N Clostridium_hylemonae*clade_260 100% 50% Y N Clostridium_scindens clade_260 10% 60% Y NCoprococcus_comes* clade_262 90% 80% Y YLachnospiraceae_bacterium_1_4_56FAA* clade_262 90% 80% Y YRuminococcus_torques clade_262 30% 70% Y Y Parabacteroides_merdaeclade_286 30% 20% N Y Bifidobacterium_bifidum clade_293 10% 0% N NJohnsonella_ignava clade_298 10% 10% N N Blautia_glucerasea* clade_309100% 80% Y N Blautia_sp_M25* clade_309 100% 70% Y YLachnospiraceae_bacterium_6_1_63FAA* clade_309 100% 60% Y NEubacterium_cellulosolvens clade_309 10% 30% Y Y Lactobacillus_fermentumclade_313 10% 0% N N Sarcina_ventriculi clade_353 10% 10% Y NClostridium_bartlettii* clade_354 90% 70% Y N Clostridium_bifermentansclade_354 70% 70% Y N Clostridium_mayombei clade_354 50% 50% Y NDorea_longicatena* clade_360 100% 60% Y YLachnospiraceae_bacterium_9_1_43BFAA clade_360 100% 30% Y NLachnospiraceae_bacterium_2_1_58FAA* clade_360 80% 80% Y NLachnospiraceae_bacterium_2_1_46FAA clade_360 50% 50% Y NLactobacillus_perolens clade_373 10% 0% N N Bacteroides_dorei clade_37860% 50% N Y Eubacterium_biforme clade_385 10% 0% Y NPeptoniphilus_sp_gpac077 clade_389 10% 20% N N Coprococcus_catus*clade_393 100% 70% Y Y Eubacterium_hallii* clade_396 90% 60% Y YAnaerosporobacter_mobilis clade_396 40% 60% Y NBacteroides_pectinophilus clade_396 10% 60% Y N Lactobacillus_hominisclade_398 10% 0% N N Lactococcus_lactis clade_401 40% 40% N NRuminococcus_champanellensis* clade_406 80% 50% Y NRuminococcus_callidus clade_406 10% 10% Y N Clostridium_clostridioforme*clade_408 100% 60% Y Y Eubacterium_hadrum* clade_408 100% 90% Y YClostridium_symbiosum clade_408 30% 50% Y Y Anaerostipes_caccaeclade_408 10% 50% Y N Parasutterella_excrementihominis clade_432 10% 0%N N Sutterella_stercoricanis clade_432 10% 0% N N Eubacterium_rectale*clade_444 100% 80% Y Y Lachnobacterium_bovis* clade_444 100% 80% Y NDesulfovibrio_desulfuricans clade_445 10% 0% N YEubacterium_sp_oral_clone_JS001* clade_476 80% 70% Y NFaecalibacterium_prausnitzii* clade_478 100% 60% Y YSubdoligranulum_variabile* clade_478 100% 80% Y Y Coprobacillus_sp_D7*clade_481 90% 60% Y N Clostridium_cocleatum clade_481 60% 20% Y NClostridium_spiroforme clade_481 40% 50% Y N Eubacterium_ramulus*clade_482 80% 60% Y N Flavonifractor_plautii clade_494 70% 60% Y YPseudoflavonifractor_capillosus clade_494 60% 60% Y YRuminococcaceae_bacterium_D16 clade_494 30% 50% Y YAcetivibrio_cellulolyticus* clade_495 70% 80% Y NClostridium_stercorarium clade_495 40% 50% Y N Enterococcus_duransclade_497 10% 10% N N Enterococcus_faecium clade_497 10% 10% N NDialister_invisus clade_506 50% 10% N N Eubacterium_limosum clade_51220% 0% Y N Ruminococcus_flavefaciens clade_516 60% 60% Y NEubacterium_ventriosum clade_519 30% 60% Y Y Bilophila_wadsworthiaclade_521 90% 0% N Y Lachnospira_pectinoschiza clade_522 40% 60% Y NEubacterium_eligens clade_522 30% 50% Y Y Catonella_morbi clade_534 20%0% N N Clostridium_sporosphaeroides* clade_537 100% 80% Y NRuminococcus_bromii clade_537 60% 30% Y Y Clostridium_leptum clade_53740% 70% Y Y Clostridium_sp_YIT_12069 clade_537 40% 60% Y NClostridium_viride clade_540 10% 10% Y N Megamonas_funiformis clade_54250% 0% N N Eubacterium_ruminantium* clade_543 80% 90% Y NCoprococcus_eutactus clade_543 20% 20% Y N Collinsella_aerofaciensclade_553 50% 10% Y Y Alkaliphilus_metalliredigenes clade_554 40% 10% YN Turicibacter_sanguinis clade_555 80% 40% Y NPhascolarctobacterium_faecium clade_556 20% 0% N NClostridiales_bacterium_oral_clone_P4PA* clade_558 80% 50% N NLutispora_thermophila clade_564 100% 0% Y NCoriobacteriaceae_bacterium_JC110 clade_566 70% 0% N NEggerthella_sp_1_3_56FAA clade_566 70% 30% N NAdlercreutzia_equolifaciens clade_566 40% 0% N N Gordonibacter_pamelaeaeclade_566 30% 0% N Y Slackia_isoflavoniconvertens clade_566 10% 0% N NEubacterium_desmolans* clade_572 90% 70% Y NPapillibacter_cinnamivorans* clade_572 90% 80% Y N Clostridium_colinumclade_576 30% 30% Y N Akkermansia_muciniphila clade_583 60% 10% N YClostridiales_bacterium_oral_taxon_F32 clade_584 60% 30% N NProchlorococcus_marinus clade_592 30% 0% N N Methanobrevibacter_woliniiclade_595 30% 0% N N Bacteroides_fragilis clade_65 20% 30% N YLactobacillus_delbrueckii clade_72 10% 0% N N Escherichia_coli clade_9250% 0% N Y Clostridium_sp_D5 clade_96 80% 60% Y NStreptococcus_thermophilus clade_98 90% 20% N Y Streptococcus_sp_CM6clade_98 20% 10% N N Streptococcus_sp_oral_clone_ASCE05 clade_98 10% 0%N N

Next, it was reasoned that for an OTU to be considered a member of theCore Ecology of the spore preparation, that OTU must be shown to engraftin a patient. Engraftment is important for two reasons. First,engraftment is a sine qua non of the mechanism to reshape the microbiomeand eliminate C. difficile colonization. OTUs that engraft with higherfrequency are highly likely to be a component of the Core Ecology of thespore preparation. Second, OTUs detected by sequencing the sporepreparation (as in Table GB) may include non-viable spores or othercontaminant DNA molecules not associated with spores. The requirementthat an OTU must be shown to engraft in the patient eliminates OTUs thatrepresent non-viable spores or contaminating sequences. Table GB alsoidentifies all OTUs detected in the spore preparation that also wereshown to engraft in at least one patient post-treatment. OTUs that arepresent in a large percentage of the ethanol spore preparations analyzedand that engraft in a large number of patients represent a subset of theCore Ecology that are highly likely to catalyze the shift from adysbiotic disease ecology to a healthy microbiome.

A third lens was applied to further refine insights into the CoreEcology of the spore preparation. Computational-based, network analysishas enabled the description of microbial ecologies that are present inthe microbiota of a broad population of healthy individuals. Thesenetwork ecologies are comprised of multiple OTUs, some of which aredefined as Keystone OTUs. Keystone OTUs are computationally defined asdescribed in Example 30. Keystone OTUs form a foundation to themicrobially ecologies in that they are found and as such are central tothe function of network ecologies in healthy subjects. Keystone OTUsassociated with microbial ecologies associated with healthy subjects areoften are missing or exist at reduced levels in subjects with disease.Keystone OTUs may exist in low, moderate, or high abundance in subjects.Table GB further notes which of the OTUs in the spore preparation areKeystone OTUs exclusively associated with individuals that are healthyand do not harbor disease.

There are several important findings from this data. A relatively smallnumber of species, 16 in total, are detected in all of the sporepreparations from 6 donors and 10 donations. This is surprising becausethe HMP database (www.hmpdacc.org) describes the enormous variability ofcommensal species across healthy individuals. The presence of a smallnumber of consistent OTUs lends support to the concept of a CoreEcology. The engraftment data further supports this conclusion. Aregression analysis shows a significant correlation between frequency ofdetection in a spore preparation and frequency of engraftment in adonor: R=0.43 (p<0.001). There is no a priori requirement that an OTUdetected frequently in the spore preparation will or should engraft. Forinstance, Lutispora thermophila, a spore former found in all ten sporepreparations, did not engraft in any of the patients. Bilophilawadsworthia, a gram negative anaerobe, is present in 9 of 10 donations,yet it does not engraft in any patient, indicating that it is likely anon-viable contaminant in the ethanol treated spore preparation.Finally, it is worth noting the high preponderance of previously definedKeystone OTUs among the most frequent OTUs in the spore preparations.

These three factors—prevalence in the spore preparation, frequency ofengraftment, and designation as a Keystone OTUs—enabled the creation ofa “Core Ecology Score” (CES) to rank individual OTUs. CES was defined asfollows:

-   -   40% weighting for presence of OTU in spore preparation    -   multiplier of 1 for presence in 1-3 spore preparations    -   multiplier of 2.5 for presence in 4-8 spore preparations    -   multiplier of 5 for presence in ≦9 spore preparations    -   40% weighting for engraftment in a patient    -   multiplier of 1 for engraftment in 1-4 patients    -   multiplier of 2.5 for engraftment in 5-6 patients    -   multiplier of 5 for engraftment in 7 patients    -   20% weighting to Keystone OTUs    -   multiplier of 1 for a Keystone OTU    -   multiplier of 0 for a non-Keystone OTU

Using this guide, the CES has a maximum possible score of 5 and aminimum possible score of 0.8. As an example, an OTU found in 8 of the10 spore preparations that engrafted in 3 patients and was a KeystoneOTU would be assigned the follow CES:

CES=(0.4×2.5)+(0.4×1)+(0.2×1)=1.6

Table GC ranks the top 20 OTUs by CES with the further requirement thatan OTU must be shown to engraft to be a considered an element of a coreecology.

TABLE GC Top 20 OTUs ranked by CES Spore Keystone OTU Clade CES FormerOTU Eubacterium_hadrum clade_408 4.2 Y Y Eubacterium_rectale clade_4444.2 Y Y Subdoligranulum_variabile clade_478 4.2 Y Y Blautia_sp_M25clade_309 4.2 Y Y Coprococcus_catus clade_393 4.2 Y YLachnospiraceae_bacterium_1_4_56FAA clade_262 4.2 Y Y Coprococcus_comesclade_262 4.2 Y Y Blautia_glucerasea clade_309 4.0 Y NLachnobacterium_bovis clade_444 4.0 Y N Clostridium_sporosphaeroidesclade_537 4.0 Y N Clostridiales_sp_SS3_4 clade_246 4.0 Y NPapillibacter_cinnamivorans clade_572 4.0 Y N Clostridium_bartlettiiclade_354 4.0 Y N Eubacterium_desmolans clade_572 4.0 Y NClostridium_clostridioforme clade_408 3.2 Y Y Dorea_longicatenaclade_360 3.2 Y Y Faecalibacterium_prausnitzii clade_478 3.2 Y YEubacterium_hallii clade_396 3.2 Y Y Clostridium_leptum clade_537 3.2 YY Lachnospiraceae_bacterium_6_1_63FAA clade_309 3.0 Y N

Example 32. Defining Efficacious Subsets of the Core Ecology

The number of organisms in the human gastrointestinal tract, as well asthe diversity between healthy individuals, is indicative of thefunctional redundancy of a healthy gut microbiome ecology (see The HumanMicrobiome Consortia. 2012. Structure, function and diversity of thehealthy human microbiome. Nature 486: 207-214). This redundancy makes ithighly likely that subsets of the Core Ecology describe therapeuticallybeneficial components of the ethanol treated spore preparation and thatsuch subsets may themselves be useful compositions for the treatment ofC. difficile infection given the ecologies functional characteristics.Using the CES, individual OTUs can be prioritized for evaluation as anefficacious subset of the Core Ecology.

Another aspect of functional redundancy is that evolutionarily relatedorganisms (i.e. those close to one another on the phylogenetic tree,e.g. those grouped into a single clade) will also be effectivesubstitutes in the Core Ecology or a subset thereof for treating C.difficile.

To one skilled in the art, the selection of appropriate OTU subsets fortesting in vitro (e.g. see Example 33 below) or in vivo (e.g. seeExamples 16 or 17) is straightforward. Subsets may be selected bypicking any 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 OTUs from TableGB, with a particular emphasis on those with higher CES, such as theOTUs described in Table GC. In addition, using the clade relationshipsdefined in Example 12 and Table 1 above, related OTUs can be selected assubstitutes for OTUs with acceptable CES values. These organisms can becultured anaerobically in vitro using the appropriate media (selectedfrom those described in Example 14 above), and then combined in adesired ratio. A typical experiment in the mouse C. difficile modelutilizes at least 10⁴ and preferably at least 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ ormore than 10⁹ colony forming units of a each microbe in the composition.Variations in the culture yields may sometimes mean that organisms arecombined in unequal ratios, e.g. 1:10, 1:100, 1:1,000, 1:10,000,1:100,000, or greater than 1:100,000. What is important in thesecompositions is that each strain be provided in a minimum amount so thatthe strain's contribution to the efficacy of the Core Ecology subset canbe measured. Using the principles and instructions described here, it isstraightforward for one of skill in the art to make clade-basedsubstitutions to test the efficacy of subsets of the Core Ecology. TableGB describes the clades for each OTU detected in a spore preparation andTable 1 describes the OTUs that can be used for substitutions based onclade relationships.

Example 33. Testing Subsets of the Core Ecology in the Mouse Model

Several subsets of the Core Ecology were tested in the C. difficilemouse model. The negative control was phosphate buffered saline and thepositive control was a 10% human fecal suspension. The subsets aredescribed in Table GD.

TABLE GD Subsets of the Core Ecology tested in the C. difficile mousemodel Substitute For Subset OTU OTU in Table 1 (Clade) Subset 1Collinsella aerofaciens none (Clade_553) Clostridium tertium C.sporogenes (Clade_252) Clostridium disporicum none (Clade_253)Clostridium innocuum Clostridium_sp_HGF2 (Clade_351) Clostridiummayombei none (Clade_354) Clostridium butyricum none (Clade_252)Coprococcus comes none (Clade_262) Clostridium hylemonae none(Clade_260) Clostridium bolteae E. hadrum (Clade_408) Clostridiumsymbiosum C. clostridioforme (Clade_408) Clostridium orbiscindensR._bacterium_D16 (Clade_494) Lachnospiraceae C. scindens (Clade_260)bacterium_5_1_57FAA Blautia producta Blautia_sp_M25 (Clade_309)Ruminococcus gnavus D. longicatena (Clade_360) Ruminococcus bromii none(Clade_537) Subset 2 Collinsella aerofaciens none (Clade_553)Clostridium butyricum none (Clade_252) Clostridium hylemonae none(Clade_260) Blautia producta Blautia_sp_M25 (Clade_309) Subset 3Collinsella aerofaciens none (Clade_553) Clostridium innocuumClostridium_sp_HGF2 (Clade_351) Coprococcus comes none (Clade_262)Ruminococcus bromii none (Clade_537) Subset 4 Clostridium butyricum none(Clade_252) Clostridium hylemonae none (Clade_260) Blautia productaBlautia_sp_M25 (Clade_309) Subset 5 Clostridium butyricum none(Clade_252) Clostridium hylemonae none (Clade_260) Subset 6 Blautiaproducta Blautia_sp_M25 (Clade_309) Clostridium butyricum none(Clade_252) Subset 7 Clostridium orbiscindens R._bacterium_D16(Clade_494) Lachnospiraceae C. scindens (Clade_260) bacterium_5_1_57FAAEubacterium rectale none (Clade_444)

Two cages of five mice each were tested for each arm of the experiment.All mice received an antibiotic cocktail consisting of 10% glucose,kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml),metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin(0.1 mg/ml) and Vancomycin (0.056 mg/ml) in their drinking water on days−14 through −5 and a dose of 10 mg/kg Clindamycin by oral gavage on day−3. On day −1, they received either the test articles or controlarticles via oral gavage. On day 0 they were challenged byadministration of approximately 4.5 log 10 cfu of C. difficile (ATCC43255) via oral gavage. Mortality was assessed every day from day 0 today 6 and the weight and subsequent weight change of the animal wasassessed with weight loss being associated with C. difficile infection.Mortality and reduced weight loss of the test article compared to theempty vehicle was used to assess the success of the test article.Additionally, a C. difficile symptom scoring was performed each day fromday −1 through day 6. Symptom scoring was based on Appearance (0-2 ptsbased on normal, hunched, piloerection, or lethargic), Respiration (0-2pts based on normal, rapid or shallow, with abdominal breathing),Clinical Signs (0-2 points based on normal, wet tail, cold-to-the-touch,or isolation from other animals).

In addition to compiling the cumulative mortality for each arm, theaverage minimum relative weight is calculated as the mean of eachmouse's minimum weight relative to Day −1 and the average maximumclinical score is calculated as the mean of each mouse's maximumcombined clinical score with a score of 4 assigned in the case of death.The results are reported in Table GE.

TABLE GE Results of bacterial compositions tested in a C. difficilemouse model. Avg. Avg. Cumulative Minimum Maximum Mortality RelativeClinical Score Group Dose (%) Weight (Death = 4) Vehicle — 40 0.87 2.8Control Feces 5.8e8 cfu total 0 0.99 0 Control Subset 1 1e8 cfu/OTU 00.98 0 Subset 2 1e8 cfu/OTU 10 0.84 2.1 Subset 3 1e8 cfu/OTU 10 0.84 2.2Subset 4 1e8 cfu/OTU 0 0.87 2 Subset 5 1e8 cfu/OTU 20 0.91 1.7 Subset 61e8 cfu/OTU 40 0.82 2.8 Subset 7 1e8 cfu/OTU 0 0.90 1

Example 34. Defining Subsets of the Core Ecology in the In Vitro C.difficile Inhibition Assay

Vials of −80° C. glycerol stock banks were thawed and diluted to 1e8CFU/mL. Selected strains and their clade assignment are given in TableGF. Each strain was then diluted 10× (to a final concentration of 1e7CFU/mL of each strain) into 200 uL of PBS+15% glycerol in the wells of a96-well plate. Plates were then frozen at −80° C. When needed for theassay, plates were removed from −80° C. and thawed at room temperatureunder anaerobic conditions when testing in a in vitro C. difficileinhibition assay (CivSim).

An overnight culture of Clostridium difficile is grown under anaerobicconditions in SweetB-Fosln or other suitable media for the growth of C.difficile. SweetB-Fosln is a complex media composed of brain heartinfusion, yeast extract, cysteine, cellobiose, maltose, soluble starch,and fructooligosaccharides/inulin, and hemin, and is buffered with MOPs.After 24 hr of growth the culture is diluted 100,000 fold into a complexmedia such as SweetB-Fosln which is suitable for the growth of a widevariety of anaerobic bacterial species. The diluted C. difficile mixtureis then aliquoted to wells of a 96-well plate (180 uL to each well). 20uL of a subset Core Ecology is then added to each well at a finalconcentration of 1e6 CFU/mL of each species. Alternatively the assay canbe tested each species at different initial concentrations (1e9 CFU/mL,1e8 CFU/mL, 1e7 CFU/mL, 1e5 CFU/mL, 1e4 CFU/mL, 1e3 CFU/mL, 1e2 CFU/mL).Control wells only inoculated with C. difficile are included for acomparison to the growth of C. difficile without inhibition. Additionalwells are used for controls that either inhibit or do not inhibit thegrowth of C. difficile. One example of a positive control that inhibitsgrowth is a combination of Blautia producta, Clostridium bifermentansand Escherichia coli. One example of a control that shows reducedinhibition of C. difficile growth is a combination of Bacteroidesthetaiotaomicron, Bacteroides ovatus and Bacteroides vulgatus. Platesare wrapped with parafilm and incubated for 24 hr at 37° C. underanaerobic conditions. After 24 hr the wells containing C. difficilealone are serially diluted and plated to determine titer. The 96-wellplate is then frozen at −80 C before quantifying C. difficile by qPCRassay.

A standard curve is generated from a well on each assay plate containingonly pathogenic C. difficile grown in SweetB+Fosln media and quantifiedby selective spot plating. Serial dilutions of the culture are performedin sterile phosphate-buffered saline. Genomic DNA is extracted from thestandard curve samples along with the other wells.

Genomic DNA is extracted from 5 μl of each sample using a dilution,freeze/thaw, and heat lysis protocol. 5 μL of thawed samples is added to45 μL of UltraPure water (Life Technologies, Carlsbad, Calif.) and mixedby pipetting. The plates with diluted samples are frozen at −20° C.until use for qPCR which includes a heated lysis step prior toamplification. Alternatively the genomic DNA is isolated using the MoBio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,Carlsbad, Calif.), Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions.

The qPCR reaction mixture contains 1× SsoAdvanced Universal ProbesSupermix, 900 nM of Wr-tcdB-F primer (AGCAGTTGAATATAGTGGTTTAGTTAGAGTTG(SEQ ID NO: 2040), IDT, Coralville, Iowa), 900 nM of Wr-tcdB-R primer(CATGCTTTTTTAGTTTCTGGATTGAA (SEQ ID NO: 2041), IDT, Coralville, Iowa),250 nM of Wr-tcdB-P probe (6FAM-CATCCAGTCTCAATTGTATATGTTTCTCCA-MGB (SEQID NO: 2042), Life Technologies, Grand Island, N.Y.), and MolecularBiology Grade Water (Mo Bio Laboratories, Carlsbad, Calif.) to 18 μl(Primers adapted from: Wroblewski, D. et al., Rapid MolecularCharacterization of Clostridium difficile and Assessment of Populationsof C. difficile in Stool Specimens, Journal of Clinical Microbiology47:2142-2148 (2009)). This reaction mixture is aliquoted to wells of aHard-shell Low-Profile Thin Wall 96-well Skirted PCR Plate (BioRad,Hercules, Calif.). To this reaction mixture, 2 μl of diluted, frozen,and thawed samples are added and the plate sealed with a Microseal ‘B’Adhesive Seal (BioRad, Hercules, Calif.). The qPCR is performed on aBioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System(BioRad, Hercules, Calif.). The thermocycling conditions are 95° C. for15 minutes followed by 45 cycles of 95° C. for 5 seconds, 60° C. for 30seconds, and fluorescent readings of the FAM channel. Alternatively, theqPCR is performed with other standard methods known to those skilled inthe art.

The Cq value for each well on the FAM channel is determined by the CFXManager™ 3.0 software. The log₁₀ (cfu/mL) of C. difficile eachexperimental sample is calculated by inputting a given sample's Cq valueinto a linear regression model generated from the standard curvecomparing the Cq values of the standard curve wells to the known log₁₀(cfu/mL) of those samples. The log inhibition is calculated for eachsample by subtracting the log₁₀ (cfu/mL) of C. difficile in the samplefrom the log₁₀ (cfu/mL) of C. difficile in the sample on each assayplate used for the generation of the standard curve that has noadditional bacteria added. The mean log inhibition is calculated for allreplicates for each composition.

A histogram of the range and standard deviation of each composition isplotted. Ranges or standard deviations of the log inhibitions that aredistinct from the overall distribution are examined as possibleoutliers. If the removal of a single log inhibition datum from one ofthe binary pairs that is identified in the histograms would bring therange or standard deviation in line with those from the majority of thesamples, that datum is removed as an outlier, and the mean loginhibition is recalculated.

The pooled variance of all samples evaluated in the assay is estimatedas the average of the sample variances weighted by the sample's degreesof freedom. The pooled standard error is then calculated as the squareroot of the pooled variance divided by the square root of the number ofsamples. Confidence intervals for the null hypothesis are determined bymultiplying the pooled standard error to the z score corresponding to agiven percentage threshold. Mean log inhibitions outside the confidenceinterval are considered to be inhibitory if positive or stimulatory ifnegative with the percent confidence corresponding to the interval used.Ternary combinations with mean log inhibition greater than 0.312 arereported as ++++ (≧99% confidence interval (C.I.) of the nullhypothesis), those with mean log inhibition between 0.221 and 0.312 as+++ (95%<C.I. <99%), those with mean log inhibition between 0.171 and0.221 as ++ (90%<C.I. <95%), those with mean log inhibition between0.113 and 0.171 as + (80%<C.I. <90%), those with mean log inhibitionbetween −0.113 and −0.171 as − (80%<C.I. <90%), those with mean loginhibition between −0.171 and −0.221 as −− (90%<C.I. <95%), those withmean log inhibition between −0.221 and −0.312 as −−− (95%<C.I. <99%),and those with mean log inhibition less than −0.312 as −−−− (99%<C.I.).

TABLE GF OTUs and their clade assignments tested in ternary combinationswith results in the in vitro inhibition assay OTU1 Clade1 OTU2 Clade2OTU3 Clade3 Results Clostridium_bolteae clade_408 Blautia_productaclade_309 Eubacterium_rectale clade_444 ++++ Clostridium_bolteaeclade_408 Clostridium_symbiosum clade_408 Blautia_producta clade_309++++ Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408Eubacterium_rectale clade_444 − Clostridium_bolteae clade_408Clostridium_symbiosum clade_408 Faecalibacterium_ clade_478 −prausnitzii Clostridium_bolteae clade_408 Clostridium_symbiosumclade_408 Lachnospiraceae_ clade_260 bacterium_5_1_57FAAClostridium_bolteae clade_408 Faecalibacterium_ clade_478Blautia_producta clade_309 ++++ prausnitzii Clostridium_bolteaeclade_408 Faecalibacterium_ clade_478 Eubacterium_rectale clade_444prausnitzii Clostridium_bolteae clade_408 Faecalibacterium_ clade_478Lachnospiraceae_ clade_260 ++++ prausnitzii bacterium_5_1_57FAAClostridium_bolteae clade_408 Lachnospiraceae_ clade_260Blautia_producta clade_309 ++++ bacterium_5_1_57FAA Clostridium_bolteaeclade_408 Lachnospiraceae_ clade_260 Eubacterium_rectale clade_444 +bacterium_5_1_57FAA Clostridium_symbiosum clade_408 Blautia_productaclade_309 Eubacterium_rectale clade_444 ++++ Clostridium_symbiosumclade_408 Faecalibacterium_ clade_478 Blautia_producta clade_309 ++++prausnitzii Clostridium_symbiosum clade_408 Faecalibacterium_ clade_478Eubacterium_rectale clade_444 prausnitzii Clostridium_symbiosumclade_408 Faecalibacterium_ clade_478 Lachnospiraceae_ clade_260 +prausnitzii bacterium_5_1_57FAA Clostridium_symbiosum clade_408Lachnospiraceae_ clade_260 Blautia_producta clade_309 ++++bacterium_5_1_57FAA Clostridium_symbiosum clade_408 Lachnospiraceae_clade_260 Eubacterium_rectale clade_444 bacterium_5_1_57FAACollinsella_aerofaciens clade_553 Blautia_producta clade_309Eubacterium_rectale clade_444 ++++ Collinsella_aerofaciens clade_553Clostridium_bolteae clade_408 Blautia_producta clade_309 ++++Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408Clostridium_symbiosum clade_408 ++++ Collinsella_aerofaciens clade_553Clostridium_bolteae clade_408 Eubacterium_rectale clade_444 ++++Collinsella_aerofaciens clade_553 Clostridium_bolteae clade_408Faecalibacterium_ clade_478 ++++ prausnitzii Collinsella_aerofaciensclade_553 Clostridium_bolteae clade_408 Lachnospiraceae_ clade_260 ++++bacterium_5_1_57FAA Collinsella_aerofaciens clade_553Clostridium_symbiosum clade_408 Blautia_producta clade_309 ++++Collinsella_aerofaciens clade_553 Clostridium_symbiosum clade_408Eubacterium_rectale clade_444 Collinsella_aerofaciens clade_553Clostridium_symbiosum clade_408 Faecalibacterium_ clade_478 prausnitziiCollinsella_aerofaciens clade_553 Clostridium_symbiosum clade_408Lachnospiraceae_ clade_260 + bacterium_5_1_57FAA Collinsella_aerofaciensclade_553 Coprococcus_comes clade_262 Blautia_producta clade_309 ++++Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262Clostridium_bolteae clade_408 ++++ Collinsella_aerofaciens clade_553Coprococcus_comes clade_262 Clostridium_symbiosum clade_408 +++Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262Eubacterium_rectale clade_444 +++ Collinsella_aerofaciens clade_553Coprococcus_comes clade_262 Faecalibacterium_ clade_478 ++++ prausnitziiCollinsella_aerofaciens clade_553 Coprococcus_comes clade_262Lachnospiraceae_ clade_260 +++ bacterium_5_1_57FAACollinsella_aerofaciens clade_553 Faecalibacterium_ clade_478Blautia_producta clade_309 ++++ prausnitzii Collinsella_aerofaciensclade_553 Faecalibacterium_ clade_478 Eubacterium_rectale clade_444 +++prausnitzii Collinsella_aerofaciens clade_553 Faecalibacterium_clade_478 Lachnospiraceae_ clade_260 +++ prausnitzii bacterium_5_1_57FAACollinsella_aerofaciens clade_553 Lachnospiraceae_ clade_260Blautia_producta clade_309 ++++ bacterium_5_1_57FAACollinsella_aerofaciens clade_553 Lachnospiraceae_ clade_260Eubacterium_rectale clade_444 ++++ bacterium_5_1_57FAA Coprococcus_comesclade_262 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++Coprococcus_comes clade_262 Clostridium_bolteae clade_408Blautia_producta clade_309 ++++ Coprococcus_comes clade_262Clostridium_bolteae clade_408 Clostridium_symbiosum clade_408Coprococcus_comes clade_262 Clostridium_bolteae clade_408Eubacterium_rectale clade_444 −− Coprococcus_comes clade_262Clostridium_bolteae clade_408 Faecalibacterium_ clade_478 +++prausnitzii Coprococcus_comes clade_262 Clostridium_bolteae clade_408Lachnospiraceae_ clade_260 +++ bacterium_5_1_57FAA Coprococcus_comesclade_262 Clostridium_symbiosum clade_408 Blautia_producta clade_309++++ Coprococcus_comes clade_262 Clostridium_symbiosum clade_408Eubacterium_rectale clade_444 --- Coprococcus_comes clade_262Clostridium_symbiosum clade_408 Faecalibacterium_ clade_478 prausnitziiCoprococcus_comes clade_262 Clostridium_symbiosum clade_408Lachnospiraceae_ clade_260 bacterium_5_1_57FAA Coprococcus_comesclade_262 Faecalibacterium_ clade_478 Blautia_producta clade_309 ++++prausnitzii Coprococcus_comes clade_262 Faecalibacterium_ clade_478Eubacterium_rectale clade_444 − prausnitzii Coprococcus_comes clade_262Faecalibacterium_ clade_478 Lachnospiraceae_ clade_260 prausnitziibacterium_5_1_57FAA Coprococcus_comes clade_262 Lachnospiraceae_clade_260 Blautia_producta clade_309 ++++ bacterium_5_1_57FAACoprococcus_comes clade_262 Lachnospiraceae_ clade_260Eubacterium_rectale clade_444 bacterium_5_1_57FAA Faecalibacterium_clade_478 Blautia_producta clade_309 Eubacterium_rectale clade_444 ++++prausnitzii Faecalibacterium_ clade_478 Lachnospiraceae_ clade_260Blautia_producta clade_309 ++++ prausnitzii bacterium_5_1_57FAAFaecalibacterium_ clade_478 Lachnospiraceae_ clade_260Eubacterium_rectale clade_444 prausnitzii bacterium_5_1_57FAALachnospiraceae_ clade_260 Blautia_producta clade_309Eubacterium_rectale clade_444 ++++ bacterium_5_1_57FAA

The CivSim shows that many ternary combinations inhibit C. difficile. 39of 56 combinations show inhibition with a confidence interval >80%; 36of 56 with a C.I. >90%; 36 of 56 with a C.I. >95%; 29 of 56 with a C.I.of >99%. Non-limiting but exemplary ternary combinations include thosewith mean log reduction greater than 0.171, e.g. any combination shownin Table 6 with a score of ++++, such as Colinsella aerofaciens,Coprococcus comes, and Blautia producta. Equally important, the CivSimassay describes ternary combinations that do not effectively inhibit C.difficile. 5 of 56 combinations promote growth with >80% confidence; 2of 56 promote growth with >90% confidence; 1 of 56, Coprococcus comes,Clostridium symbiosum and Eubacterium rectale, promote growth with >95%confidence. 12 of 56 combinations are neutral in the assay, meaning theyneither promote nor inhibit C. difficile growth to the limit ofmeasurement.

It is straightforward for one of skill in the art to use the CivSimmethod to determine efficacious subsets of the Core Ecology derived fromthe ethanol treated spore fraction shown to be efficacious in treatingC. difficile in humans.

Example AAZA: Bacterial Compositions Populating the Gut in a Mouse Model

Two bacterial compositions were evaluated in a mouse model todemonstrate the ability to populate the gastrointestinal tract. Bacteriawere grown as described in ***Example 14. Compositions were pre-madeunder anaerobic conditions and suspended in PBS+15% glycerol and storedat ≦−70° C. prior to

Groups of mice (10 females/group; 5 per cage) were pre-treated on Days−14 to −5 with an antibiotic cocktail consisting of 10% glucose,kanamycin (0.5 mg/ml), gentamicin (0.044 mg/ml), colistin (1062.5 U/ml),metronidazole (0.269 mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin(0.1 mg/ml) and vancomycin (0.056 mg/ml) in their drinking water. On Day−3 they received 10 mg/kg Clindamycin by oral gavage. On Day −1, theywere dosed with a microbial compositions by oral gavage in a volume of0.2 mL (Table ZA). Microbial compositions comprised approximately equalnumbers of each OTU and were dosed at approximately 1×10⁹, 1×10⁸ and1×10⁷ per OTU for each composition (e.g. microbial composition 1,comprising 15 strains, was dosed at approximately 1.5×10¹⁰, 1.5×10⁹, and1.5×10⁸ total CFU). Fecal samples were collected from each cage on Day−1 (approximately 1 hour before dosing) and on Days 2, 3 and 4post-dosing. Feces were stored frozen prior to processing andsequencing. Weight gain of mice treated with either microbialcompositions was similar to that of naive, control mice.

In parallel, groups of animals treated with the same microbialcompositions on Day −1 were challenged on Day 0 with approximately10^(4.5) spores of Clostridium difficile (ATCC 43255) via oral gavage.Mortality for C. difficile challenged animals was assessed every dayfrom Day 0 to Day 6 and the weight and subsequent weight change of theanimal was assessed with weight loss being associated with C. difficileinfection. Mortality and reduced weight loss of the test articlecompared to the empty vehicle was used to assess the success of the testarticle.

TABLE ZA Microbial compositions administered via oral gavage on Day −1OTU Clade Microbial Clostridium_butyricum clade_252 Composition 1Clostridium_disporicum clade_253 Clostridium_hylemonae clade_260Clostridium_orbiscindens clade_494 Clostridium_symbiosum clade_408Collinsella_aerofaciens clade_553 Coprococcus_comes clade_262Lachnospiraceae_bacterium_5_1_57FAA clade_260 Ruminococcus_bromiiclade_537 Blautia_producta clade_309 Clostridium_bolteae clade_408Clostridium_innocuum clade_351 Clostridium_mayombei clade_354Clostridium_tertium clade_252 Ruminococcus_gnavus clade_360 MicrobialClostridium_disporicum clade_253 Composition 2 Clostridium_orbiscindensclade_494 Clostridium_symbiosum clade_408 Collinsella_aerofaciensclade_553 Eubacterium_rectale clade_444Lachnospiraceae_bacterium_5_1_57FAA clade_260 Blautia_producta clade_309Clostridium_innocuum clade_351 Clostridium_mayombei clade_354

Fecal samples were processed by isolating and sequencing DNA accordingto ***Example 11 and 12. The OTU assignment of fecal samples from Days−1, 2, 3 and 4 was determined by analyzing 16S-V4 sequence reads andassigning OTUs as described in ***Example 11. clades were assigned asdescribed in ***Example 11. Total read counts were determined for eachOTU or each clade by summing the results from cages of the sameexperimental group. Samples with 10 or fewer sequence reads for a givenOTU or clade were considered to be below background and were notincluded in the summation process. Results are shown by OTU (Table TAB)and by clade (Table TAC).

TABLE TAB Population of OTUs on Days 2, 3 and 4 following dosing withMicrobial Compositions 1 × 10⁹ per OTU 1 × 10⁸ per OTU 1 × 10⁷ per OTU−1 2 3 4 −1 2 3 4 −1 2 3 4 Microbial comp 1 Cl_butyricum 0 106 51 32 010 0 34 195 0 0 0 Cl_disporicum 10 1746 1190 887 0 1746 769 1011 20111175 1531 1152 Cl_hylemonae 0 258 258 84 0 203 164 77 0 265 214 90Cl_orbiscindens 0 188 192 471 0 188 138 276 0 221 174 341 Cl_symbiosum 0485 482 486 0 444 379 447 0 562 427 775 Co_aerofaciens 0 0 0 0 0 0 0 0 00 0 0 C_comes 0 0 0 0 0 0 0 0 0 0 0 0 L_bacterium_5_1_57FAA 0 341 336354 0 351 182 356 0 256 240 300 R_bromii 0 0 0 0 0 0 0 0 0 0 0 0B_producta 0 0 0 0 0 0 0 0 0 0 0 0 Cl_bolteae 0 0 0 0 0 0 0 0 0 0 0 0Cl_innocuum 0 0 0 0 0 0 0 0 0 0 0 0 Cl_mayombei 0 0 0 0 0 0 0 0 0 0 0 0Cl_tertium 0 0 0 0 0 0 0 0 0 0 0 0 R_gnavus 0 0 0 0 0 0 0 0 0 0 0 0Microbial comp 2 Cl_disporicum 29 11810 10948 14672 0 11349 13978 3942 011995 7005 6268 Cl_orbiscindens 0 510 408 764 0 332 545 544 0 310 319432 Cl_symbiosum 0 559 508 375 0 665 494 450 0 396 639 650Co_aerofaciens 0 0 0 0 0 0 1172 0 0 0 247 0 E_rectale 0 0 0 0 0 0 0 12 00 0 261 L_bacterium_5_1_57FAA 0 972 801 596 0 860 962 844 0 636 19011269 B_producta 0 0 0 0 0 0 0 0 0 0 0 0 Cl_innocuum 0 0 0 0 0 0 0 0 0 00 0 Cl_mayombei 0 0 0 0 0 0 0 0 0 0 0 0

TABLE TAC Population of Clades on Days 2, 3 and 4 following dosing withMicrobial Compositions 1 × 10⁹ per OTU 1 × 10⁸ per OTU 1 × 10⁷ per OTU−1 2 3 4 −1 2 3 4 −1 2 3 4 Microbial comp 1 clade_252 0 444 252 87 0 198122 125 209 394 231 88 clade_253 10 1746 1190 887 0 1746 769 1011 20111175 1531 1152 clade_260 0 599 594 438 0 554 346 433 0 521 454 390clade_262 0 14 151 51 0 0 0 0 0 12 21 57 clade_309 0 11093 9750 4023 09991 5208 5145 19 9311 6369 4951 clade_351 0 9064 10647 7751 0 6528 72598213 0 8903 10049 8701 clade_354 0 0 0 0 0 0 0 31 173 0 0 0 clade_360 014300 10220 11036 0 12553 12989 6889 0 9308 13483 9292 clade_408 13 889212985 12101 23 3952 7260 10652 43 4079 8581 14929 clade_494 0 226 227565 0 188 184 411 0 221 200 351 clade_537 0 0 68 225 0 0 0 0 0 0 0 55clade_553 0 0 0 0 0 0 0 0 0 0 0 0 Microbial comp 2 clade_253 29 1181010948 14672 0 11349 13978 3942 0 11995 7005 6268 clade_260 0 1125 1312854 0 1049 1295 1250 0 792 2121 1637 clade_309 54 12513 13731 7849 011610 12004 12672 0 7407 14111 10858 clade_351 0 7651 9939 5936 0 84959724 9207 0 6005 9833 7655 clade_354 149 0 127 429 0 0 0 39 12 0 0 0clade_408 18 2242 4989 10480 12 1688 5580 3789 0 1068 1561 6281clade_444 41 0 49 202 0 18 0 12 0 14 82 1578 clade_494 0 510 465 1054 0332 565 596 0 310 319 476 clade_553 0 0 0 0 0 0 1172 0 0 0 247 0

Upon examining the OTU data in Table TAB, several patterns emerge.First, there are a group of OTUs with no sequence reads on Day −1 thatshow subsequent and large numbers of sequence reads on Days 2, 3, or 4;this group includes Cl. butyricum, Cl. hylemonae, Cl. orbiscindens, Cl.symbiosum, and L. bacterium_5_1_57 FAA. Cl. disporicum is comparable tothis group as it has sequence reads on Day −1 that are very close tobackground (10 and 29 in compositions 1 and 2, respectively), whichsubsequently increase by as much as 1000-fold on Days 2, 3 or 4. Second,there are OTUs such as Co. aerofaciens, C. comes, R. bromii, B.producta, Cl. bolteae, Cl. mayombei, Cl. innocuum, Cl. tertium and R.gnavus which are not detectable at the OTU level in either the Day −1sample or in subsequent samples. In composition 2, Co. aerofaciens isdetected transiently on Day 2 in the 1×10⁸ and 1×10⁷ dose groups; E.rectale in the same experimental groups is detected on Day 3, suggestinga possible relationship between transient population by Co. aerofaciensfollowed by E. rectale in these groups of mice. A striking observationis that the observed number of OTU sequence reads is not highly dosedependent. Overall, the data is consistent with a model whereby OTUspopulate rapidly following oral administration.

The clade-based analysis in Table TAC was performed to more thoroughlyevaluate the population of the GI tract. Clade-based analysis obscuressome of the details afforded by an OTU analysis. For instance, Cl.tertium and Cl. butyricum are members of the same clade and thus aclade-based analysis cannot distinguish the dynamics of these individualOTUs. However, clade-based analysis has the compensatory benefit that itis sensitive to measuring population changes that can be missed by anOTU-based analysis. The ability of 16S-V4 OTU identification to assignan OTU as a specific species depends in part on the resolving power ofthe 16S-V4 region for a particular species or group of species. Both thedensity of available reference 16S sequences for different regions ofthe tree as well as the inherent variability in the 16S gene betweendifferent species will determine the definitiveness of a taxonomicannotation. So in some cases, the population of a species can befollowed using clade-based assignments when OTU based-detection isinsensitive in a complex population. For instance, the clade-basedanalysis in Table 2B supports the case that R. bromii, B. producta, Cl.innocuum, and R. gnavus were able to populate since each OTU is a solemember of a clade in the microbial compositions and sequence reads wentfrom undetectable on Day −1 to well above background on Days 2, 3 or 4.16S V4 sequencing and clade-based analysis could not determine whetherCl. tertium or Cl. bolteae populated due to the fact that other membersof their clades (Cl. butyricum and Cl. symbiosum, respectively) werepresent and shown to populate at the OTU level in the mice.

In the mice challenged in parallel with C. difficile, animals weresignificantly protected as shown in Table TAD. Mice gavaged with vehicle(phosphate buffered saline) experienced 100% mortality while microbialcompositions 1 and 2 protected at all dose levels with between 0 and 10%mortality by Day 6, the last day of the experiment. In addition, weightloss in animals treated with microbial compositions 1 and 2 was minimalcompared to animals receiving the vehicle gavage. These data confirmthat population of the gastrointestinal tract with microbialcompositions confers a clinical benefit by restoring a state ofdysbiosis so that animals can resist infection by a pathogen.

TABLE TAD Mortality by experimental group in mice challenged with10^(4.5) C. difficile spores on Day 0 Group Dose (CFU per OTU) Deaths (%mortality) Vehicle control N/A 10 (100%) Microbial composition 1 109 1(10%) 108 1 (10%) 107 0 (0%)  Microbial composition 2 109 0 (0%)  108 1(10%) 107 1 (10%)

Example 36: Prophylactic Use and Treatment in a Mouse Model ofVancomycin Resistant Enterococcus (VRE) Colonization

The emergence and spread of highly antibiotic-resistant bacteriarepresent a major clinical challenge (Snitkin et al ScienceTranslational Medicine, 2012). In recent years, the numbers ofinfections caused by organisms such as methicillin-resistantStaphylococcus aureus, carbapenem-resistant Enterobacteriaceae,vancomycin-resistant Enterococcus (VRE), and Clostridium difficile haveincreased markedly, and many of these strains are acquiring resistanceto the few remaining active antibiotics. Most infections produced byhighly antibiotic-resistant bacteria are acquired duringhospitalizations, and preventing patient-to-patient transmission ofthese pathogens is one of the major challenges confronting hospitals andclinics. Most highly antibiotic-resistant bacterial strains belong togenera that colonize mucosal surfaces, usually at low densities. Thehighly complex microbiota that normally colonizes mucosal surfacesinhibits expansion of and domination by bacteria such asEnterobacteriaceae and Enterococcaceae. Destruction of the normal floraby antibiotic administration, however, disinhibitionantibiotic-resistant members of these bacterial families, leading totheir expansion to very high densities (Ubeda et al Journal of ClinicalInvestigation 2010). High-density colonization by these organisms can becalamitous for the susceptible patient, resulting in bacteremia andsepsis (Taur et al, Clinical Infectious Disease, 2012).

To test prophylactic use and treatment of a bacterial composition testarticle e.g. spore population, a VRE infection mouse model is used aspreviously described (Ubeda et al, Infectious Immunity 2013, Ubeda etal, Journal of clinical investigation, 2010). Briefly, experiments aredone with 7-week-old C57BL/6J female mice purchased from JacksonLaboratory, housed with irradiated food, and provided with acidifiedwater. Mice are individually housed to avoid contamination between micedue to coprophagia. For experimental infections with VRE, mice aretreated with ampicillin (0.5 g/liter) in their drinking water, which ischanged every 3 days.

In the treatment model, on day 1, mice are infected by means of oralgavage with 10⁸ CFU of the vancomycin-resistant Enterococcus faeciumstrain purchased from ATCC (ATCC 700221). One day after infection (day1), antibiotic treatment is stopped and VRE levels are determined atdifferent time points by plating serial dilutions of fecal pellets onEnterococcosel agar plates (Difco) with vancomycin (8 ug/ml; Sigma). VREcolonies are identified by appearance and confirmed by Gram staining orother methods previously described (e.g. see example 1, 2 and 3). Inaddition, as previously described (Ubeda et al Journal of ClinicalInvestigation 2010), PCR of the vanA gene, which confers resistance tovancomycin, confirms the presence of VRE in infected mice. The testarticle e.g. bacterial composition, or ethanol treated, gradientpurified spore preparation (as described herein), fecal suspension, orantibiotic treatment is delivered in PBS on days 1-3 while the negativecontrol contains only PBS and is also delivered on days 1-3 by oralgavage. Fresh fecal stool pellets are obtained daily for the duration ofthe experiment from days −7 to day 10. The samples are immediatelyfrozen and stored at −80° C. DNA was extracted using standard techniquesand analyzed with 16S or comparable methods (e.g. see example 2 and 3).

In the colonization model, ampicillin is administered as described abovefor day −7 to day 1, treatment with the test article or vehicle controlis administered on day 0-2 and the VRE resistant bacteria at 10⁸ CFU areadministered on day 14. Fecal samples are taken throughout theexperiment daily from −7 to day 21 and submitted for 16S sequencing aspreviously described (e.g. see examples 2 and 3).

In both models titers of VRE in feces are used to evaluate the successof the test article versus the negative control. Furthermore, microbiotacomposition is assessed for the ability of the test article to induce ahealthy microbiome.

Example 37: Prophylactic Use and Treatment of a Mouse Model ofCarbapenem Resistant Klebsiella (CRKB) Colonization

The emergence of Klebsiella pneumoniae strains with decreasedsusceptibility to carbapenems is a significant threat to hospitalizedpatients. Resistance to carbapenems in these organisms is mostfrequently mediated by K. pneumoniae carbapenemase (KPC), a class Abeta-lactamase that also confers resistance to broad-spectrumcephalosporins and commercially available beta-lactam/beta-lactamaseinhibitor combinations (Queenan et al, Clinical Microbiology Review,2007). KPC-producing K. pneumoniae (KPC-Kp) strains often harborresistance determinants against several other classes of antimicrobials,including aminoglycosides and fluoroquinolones, resulting in trulymultidrug-resistant (MDR) organisms (Hirsch et al, Journal ofAntimicrobial Chemotherapy, 2009). Considering the limited antimicrobialoptions, infections caused by KPC-Kp pose a tremendous therapeuticchallenge and are associated with poor clinical outcomes

A treatment protocol in a mouse model as previously described (e.g.Perez et al, Antimicrobial Agents Chemotherapy, 2011) is used toevaluate the test article e.g. bacterial composition for treatingcarbapenem resistant Klebsiella and reducing carriage in the GI tract.Female CF1 mice (Harlan Sprague-Dawley, Indianapolis, Ind.) are used andare individually housed and weighed between 25 and 30 g.

The thoroughly characterized strain of K. pneumoniae, VA-367 (8, 9, 25)is used in this study. This clinical isolate is genetically related tothe KPC-Kp strain circulating in the Eastern United States.Characterization of the resistance mechanisms in K. pneumoniae VA-367with PCR and DNA sequence analysis revealed the presence of bla_(KPC-3),bla_(SHV-11), and bla_(SHV-12) as well as qnrB19 and aac(6′)-lb.Additionally, PCR and DNA sequencing revealed disruptions in the codingsequences of the following outer membrane protein genes: ompK35, ompK36,and ompK37. Antibiotic susceptibility testing (AST) was performed withthe agar dilution method and interpreted according to currentrecommendations from the Clinical and Laboratory Standards Institute(CLSI). A modified Hodge test was performed, according to a methoddescribed previously (e.g. see Anderson et al, Journal of ClinicalMicrobiology, 2007) with ertapenem, meropenem, and imipenem. Tigecyclineand polymyxin E were evaluated by Etest susceptibility assays (AB bioMerieux, Solna, Sweden). Results for tigecycline were interpreted assuggested by the U.S. Food and Drug Administration (FDA) and accordingto CLSI recommendations (criteria for Pseudomonas) for polymyxin E.

Mice (10 per group) are assigned to either a test article e.g. bacterialcomposition, ethanol treated, spore preparation (e.g. see example 6),antibiotic clindamycin, piperacillin-tazobactam, tigecycline, ertapenem,cefepime, ciprofloxacin, or combination thereof or control groupreceiving only the vehicle. They are administered the test article dailyfrom day −10 to day 0, On day 0, 10³ CFU of KPC-Kp VA-367 diluted in 0.5ml phosphate-buffered saline (PBS) was administered by oral gavage usinga stainless-steel feeding tube (Perfektum, Popper & Sons, New Hyde Park,N.Y.). Stool samples were collected 1, 4, 6, and 11 days after theadministration of KPC-Kp in order to measure the concentration ofcarbapenem-resistant K. pneumoniae. Stool samples (100 mg diluted in 800ml of PBS) are plated onto MacConkey agar with and without 0.5 ug/ml ofimipenem, and the number of CFU per gram of stool was determined.Alternatively other methods may be used to measure the levels ofcarbapenem-resistant K. pneumoniae e.g. per, antigen testing, as onewho's skilled in the art could perform.

Stool samples were collected after 5 days of treatment to assess theeffects of the antibiotics on the stool microflora and to measureantibiotic levels in stool. To assess the effects on the microflora,fresh stool samples as previously described (e.g. see examples 2 and 3).Additional experiments are performed to examine whether theadministration the test article e.g. bacterial composition resulted inthe elimination or persistence of colonization with KPC-Kp VA-367.

Mice are treated with subcutaneous clindamycin to reduce the normalintestinal flora 1 day before receiving 10⁴ CFU of KPC-Kp VA-367 by oralgavage, and the mice continued to receive subcutaneous clindamycin everyother day for 7 days. Concurrently, for 7 days after oral gavage withKPC-Kp, mice received oral gavage of normal saline (control group), orthe bacterial composition as specified. An additional dose ofsubcutaneous clindamycin was administered 20 days after theadministration of KPC-Kp VA-367 to assess whether low levels ofcarbapenem-resistant K. pneumoniae were present that could be augmentedby the elimination of the anaerobic microflora. Stool samples werecollected at baseline and at 3, 6, 8, 11, 16, and 21 days after KPC-KpVA-367 was given by gavage. The bacterial composition will be examinedby the reduction of CRKB in feces.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in thespecification, including claims, are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless otherwiseindicated to the contrary, the numerical parameters are approximationsand may vary depending upon the desired properties sought to beobtained. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

Additional Tables

TABLE 1 List of Operational Taxonomic Units (OTU) with taxonomicassignments made to Genus, Species, and Phylogenetic Clade Clademembership of bacterial OTUs is based on 16S sequence data. Clades aredefined based on the topology of a phylogenetic tree that is constructedfrom full-length 16S sequences using maximum likelihood methods familiarto individuals with ordinary skill in the art of phylogenetics. Cladesare constructed to ensure that all OTUs in a given clade are: (i) withina specified number of bootstrap supported nodes from one another, and(ii) within 5% genetic similarity. OTUs that are within the same cladecan be distinguished as genetically and phylogenetically distinct fromOTUs in a different clade based on 16S-V4 sequence data, while OTUsfalling within the same clade are closely related. OTUs falling withinthe same clade are evolutionarily closely related and may or may not bedistinguishable from one another using 16S-V4 sequence data. Members ofthe same clade, due to their evolutionary relatedness, play similarfunctional roles in a microbial ecology such as that found in the humangut. Compositions substituting one species with another from the sameclade are likely to have conserved ecological function and therefore areuseful in the present invention. All OTUs are denoted as to theirputative capacity to form spores and whether they are a Pathogen orPathobiont (see Definitions for description of “Pathobiont”). NIAIDPriority Pathogens are denoted as ‘Category-A’, ‘Category-B’, or‘Category-C’, and Opportunistic Pathogens are denoted as ‘OP’. OTUs thatare not pathogenic or for which their ability to exist as a pathogen isunknown are denoted as ‘N’. The ‘SEQ ID Number’ denotes the identifierof the OTU in the Sequence Listing File and ‘Public DB Accession’denotes the identifier of the OTU in a public sequence repository. SEQID Public DB Spore Pathogen OTU Number Accession Clade Former StatusEubacterium saburreum 858 AB525414 clade_178 Y N Eubacterium sp. oralclone 866 AY349376 clade_178 Y N IR009 Lachnospiraceae bacterium 1061HQ616401 clade_178 Y N ICM62 Lachnospiraceae bacterium 1062 HQ616384clade_178 Y N MSX33 Lachnospiraceae bacterium 1063 ADDS01000069clade_178 Y N oral taxon 107 Alicyclobacillus acidocaldarius 122NR_074721 clade_179 Y N Clostridium baratii 555 NR_029229 clade_223 Y NClostridium colicanis 576 FJ957863 clade_223 Y N Clostridiumparaputrificum 611 AB536771 clade_223 Y N Clostridium sardiniense 621NR_041006 clade_223 Y N Eubacterium budayi 837 NR_024682 clade_223 Y NEubacterium moniliforme 851 HF558373 clade_223 Y N Eubacteriummultiforme 852 NR_024683 clade_223 Y N Eubacterium nitritogenes 853NR_024684 clade_223 Y N Anoxybacillus flavithermus 173 NR_074667clade_238 Y N Bacillus aerophilus 196 NR_042339 clade_238 Y N Bacillusaestuarii 197 GQ980243 clade_238 Y N Bacillus amyloliquefaciens 199NR_075005 clade_238 Y N Bacillus anthracis 200 AAEN01000020 clade_238 YCategory-A Bacillus atrophaeus 201 NR_075016 clade_238 Y OP Bacillusbadius 202 NR_036893 clade_238 Y OP Bacillus cereus 203 ABDJ01000015clade_238 Y OP Bacillus circulans 204 AB271747 clade_238 Y OP Bacillusfirmus 207 NR_025842 clade_238 Y OP Bacillus flexus 208 NR_024691clade_238 Y OP Bacillus fordii 209 NR_025786 clade_238 Y OP Bacillushalmapalus 211 NR_026144 clade_238 Y OP Bacillus herbersteinensis 213NR_042286 clade_238 Y OP Bacillus idriensis 215 NR_043268 clade_238 Y OPBacillus lentus 216 NR_040792 clade_238 Y OP Bacillus licheniformis 217NC_006270 clade_238 Y OP Bacillus megaterium 218 GU252124 clade_238 Y OPBacillus nealsonii 219 NR_044546 clade_238 Y OP Bacillus niabensis 220NR_043334 clade_238 Y OP Bacillus niacini 221 NR_024695 clade_238 Y OPBacillus pocheonensis 222 NR_041377 clade_238 Y OP Bacillus pumilus 223NR_074977 clade_238 Y OP Bacillus safensis 224 JQ624766 clade_238 Y OPBacillus simplex 225 NR_042136 clade_238 Y OP Bacillus sonorensis 226NR_025130 clade_238 Y OP Bacillus sp. 10403023 227 CAET01000089clade_238 Y OP MM10403188 Bacillus sp. 2_A_57_CT2 230 ACWD01000095clade_238 Y OP Bacillus sp. 2008724126 228 GU252108 clade_238 Y OPBacillus sp. 2008724139 229 GU252111 clade_238 Y OP Bacillus sp. 7_16AIA231 FN397518 clade_238 Y OP Bacillus sp. AP8 233 JX101689 clade_238 Y OPBacillus sp. B27(2008) 234 EU362173 clade_238 Y OP Bacillus sp. BT1B_CT2235 ACWC01000034 clade_238 Y OP Bacillus sp. GB1.1 236 FJ897765clade_238 Y OP Bacillus sp. GB9 237 FJ897766 clade_238 Y OP Bacillus sp.HU19.1 238 FJ897769 clade_238 Y OP Bacillus sp. HU29 239 FJ897771clade_238 Y OP Bacillus sp. HU33.1 240 FJ897772 clade_238 Y OP Bacillussp. JC6 241 JF824800 clade_238 Y OP Bacillus sp. oral taxon F79 248HM099654 clade_238 Y OP Bacillus sp. SRC_DSF1 243 GU797283 clade_238 YOP Bacillus sp. SRC_DSF10 242 GU797292 clade_238 Y OP Bacillus sp.SRC_DSF2 244 GU797284 clade_238 Y OP Bacillus sp. SRC_DSF6 245 GU797288clade_238 Y OP Bacillus sp. tc09 249 HQ844242 clade_238 Y OP Bacillussp. zh168 250 FJ851424 clade_238 Y OP Bacillus sphaericus 251 DQ286318clade_238 Y OP Bacillus sporothermodurans 252 NR_026010 clade_238 Y OPBacillus subtilis 253 EU627588 clade_238 Y OP Bacillus thermoamylovorans254 NR_029151 clade_238 Y OP Bacillus thuringiensis 255 NC_008600clade_238 Y OP Bacillus weihenstephanensis 256 NR_074926 clade_238 Y OPGeobacillus kaustophilus 933 NR_074989 clade_238 Y N Geobacillus 936NR_040794 clade_238 Y N stearothermophilus Geobacillus 938 NR_074976clade_238 Y N thermodenitrificans Geobacillus 939 NR_043022 clade_238 YN thermoglucosidasius Lysinibacillus sphaericus 1193 NR_074883 clade_238Y N Clostridiales sp. SS3_4 543 AY305316 clade_246 Y N Clostridiumbeijerinckii 557 NR_074434 clade_252 Y N Clostridium botulinum 560NC_010723 clade_252 Y Category-A Clostridium butyricum 561 ABDT01000017clade_252 Y N Clostridium chauvoei 568 EU106372 clade_252 Y NClostridium favososporum 582 X76749 clade_252 Y N Clostridiumhistolyticum 592 HF558362 clade_252 Y N Clostridium isatidis 597NR_026347 clade_252 Y N Clostridium limosum 602 FR870444 clade_252 Y NClostridium sartagoforme 622 NR_026490 clade_252 Y N Clostridiumsepticum 624 NR_026020 clade_252 Y N Clostridium sp. 7_2_43FAA 626ACDK01000101 clade_252 Y N Clostridium sporogenes 645 ABKW02000003clade_252 Y N Clostridium tertium 653 Y18174 clade_252 Y N Clostridiumcarnis 564 NR_044716 clade_253 Y N Clostridium celatum 565 X77844clade_253 Y N Clostridium disporicum 579 NR_026491 clade_253 Y NClostridium gasigenes 585 NR_024945 clade_253 Y N Clostridium quinii 616NR_026149 clade_253 Y N Clostridium hylemonae 593 AB023973 clade_260 Y NClostridium scindens 623 AF262238 clade_260 Y N Lachnospiraceaebacterium 1054 ACTR01000020 clade_260 Y N 5_1_57FAA Clostridiumglycyrrhizinilyticum 588 AB233029 clade_262 Y N Clostridium nexile 607X73443 clade_262 Y N Coprococcus comes 674 ABVR01000038 clade_262 Y NLachnospiraceae bacterium 1048 ACTM01000065 clade_262 Y N 1_1_57FAALachnospiraceae bacterium 1049 ACTN01000028 clade_262 Y N 1_4_56FAALachnospiraceae bacterium 1057 ACWQ01000079 clade_262 Y N 8_1_57FAARuminococcus lactaris 1663 ABOU02000049 clade_262 Y N Ruminococcustorques 1670 AAVP02000002 clade_262 Y N Paenibacillus lautus 1397NR_040882 clade_270 Y N Paenibacillus polymyxa 1399 NR_037006 clade_270Y N Paenibacillus sp. HGF5 1402 AEXS01000095 clade_270 Y N Paenibacillussp. HGF7 1403 AFDH01000147 clade_270 Y N Eubacterium sp. oral clone 868AY349379 clade_298 Y N JI012 Alicyclobacillus contaminans 124 NR_041475clade_301 Y N Alicyclobacillus herbarius 126 NR_024753 clade_301 Y NAlicyclobacillus pomorum 127 NR_024801 clade_301 Y N Blautia coccoides373 AB571656 clade_309 Y N Blautia glucerasea 374 AB588023 clade_309 Y NBlautia glucerasei 375 AB439724 clade_309 Y N Blautia hansenii 376ABYU02000037 clade_309 Y N Blautia luti 378 AB691576 clade_309 Y NBlautia producta 379 AB600998 clade_309 Y N Blautia schinkii 380NR_026312 clade_309 Y N Blautia sp. M25 381 HM626178 clade_309 Y NBlautia stercoris 382 HM626177 clade_309 Y N Blautia wexlerae 383EF036467 clade_309 Y N Bryantella formatexigens 439 ACCL02000018clade_309 Y N Clostridium coccoides 573 EF025906 clade_309 Y NEubacterium cellulosolvens 839 AY178842 clade_309 Y N Lachnospiraceaebacterium 1056 ACTV01000014 clade_309 Y N 6_1_63FAA Ruminococcushansenii 1662 M59114 clade_309 Y N Ruminococcus obeum 1664 AY169419clade_309 Y N Ruminococcus sp. 1666 ACII01000172 clade_309 Y N5_1_39BFAA Ruminococcus sp. K_1 1669 AB222208 clade_309 Y NSyntrophococcus sucromutans 1911 NR_036869 clade_309 Y N Bacillusalcalophilus 198 X76436 clade_327 Y N Bacillus clausii 205 FN397477clade_327 Y OP Bacillus gelatini 210 NR_025595 clade_327 Y OP Bacillushalodurans 212 AY144582 clade_327 Y OP Bacillus sp. oral taxon F26 246HM099642 clade_327 Y OP Clostridium innocuum 595 M23732 clade_351 Y NClostridium sp. HGF2 628 AENW01000022 clade_351 Y N Clostridiumperfringens 612 ABDW01000023 clade_353 Y Category-B Sarcina ventriculi1687 NR_026146 clade_353 Y N Clostridium bartlettii 556 ABEZ02000012clade_354 Y N Clostridium bifermentans 558 X73437 clade_354 Y NClostridium ghonii 586 AB542933 clade_354 Y N Clostridium glycolicum 587FJ384385 clade_354 Y N Clostridium mayombei 605 FR733682 clade_354 Y NClostridium sordellii 625 AB448946 clade_354 Y N Clostridium sp. MT4 E635 FJ159523 clade_354 Y N Eubacterium tenue 872 M59118 clade_354 Y NClostridium argentinense 553 NR_029232 clade_355 Y N Clostridium sp.JC122 630 CAEV01000127 clade_355 Y N Clostridium sp. NMBHI_1 636JN093130 clade_355 Y N Clostridium subterminale 650 NR_041795 clade_355Y N Clostridium sulfidigenes 651 NR_044161 clade_355 Y N Doreaformicigenerans 773 AAXA02000006 clade_360 Y N Dorea longicatena 774AJ132842 clade_360 Y N Lachnospiraceae bacterium 1050 ADLB01000035clade_360 Y N 2_1_46FAA Lachnospiraceae bacterium 1051 ACTO01000052clade_360 Y N 2_1_58FAA Lachnospiraceae bacterium 1053 ADCR01000030clade_360 Y N 4_1_37FAA Lachnospiraceae bacterium 1058 ACTX01000023clade_360 Y N 9_1_43BFAA Ruminococcus gnavus 1661 X94967 clade_360 Y NRuminococcus sp. ID8 1668 AY960564 clade_360 Y N Blautiahydrogenotrophica 377 ACBZ01000217 clade_368 Y N Lactonifactorlongoviformis 1147 DQ100449 clade_368 Y N Robinsoniella peoriensis 1633AF445258 clade_368 Y N Eubacterium infirmum 849 U13039 clade_384 Y NEubacterium sp. WAL 14571 864 FJ687606 clade_384 Y N Erysipelotrichaceaebacterium 823 ACZW01000054 clade_385 Y N 5_2_54FAA Eubacterium biforme835 ABYT01000002 clade_385 Y N Eubacterium cylindroides 842 FP929041clade_385 Y N Eubacterium dolichum 844 L34682 clade_385 Y N Eubacteriumsp. 3_1_31 861 ACTL01000045 clade_385 Y N Eubacterium tortuosum 873NR_044648 clade_385 Y N Bulleidia extructa 441 ADFR01000011 clade_388 YN Solobacterium moorei 1739 AECQ01000039 clade_388 Y N Coprococcus catus673 EU266552 clade_393 Y N Lachnospiraceae bacterium 1064 HM099641clade_393 Y N oral taxon F15 Clostridium cochlearium 574 NR_044717clade_395 Y N Clostridium malenominatum 604 FR749893 clade_395 Y NClostridium tetani 654 NC_004557 clade_395 Y N Acetivibrioethanolgignens 6 FR749897 clade_396 Y N Anaerosporobacter mobilis 161NR_042953 clade_396 Y N Bacteroides pectinophilus 288 ABVQ01000036clade_396 Y N Clostridium aminovalericum 551 NR_029245 clade_396 Y NClostridium phytofermentans 613 NR_074652 clade_396 Y N Eubacteriumhallii 848 L34621 clade_396 Y N Eubacterium xylanophilum 875 L34628clade_396 Y N Ruminococcus callidus 1658 NR_029160 clade_406 Y NRuminococcus 1659 FP929052 clade_406 Y N champanellensis Ruminococcussp. 18P13 1665 AJ515913 clade_406 Y N Ruminococcus sp. 9SE51 1667FM954974 clade_406 Y N Anaerostipes caccae 162 ABAX03000023 clade_408 YN Anaerostipes sp. 3_2_56FAA 163 ACWB01000002 clade_408 Y NClostridiales bacterium 541 ABQR01000074 clade_408 Y N 1_7_47FAAClostridiales sp. SM4_1 542 FP929060 clade_408 Y N Clostridiales sp.SSC_2 544 FP929061 clade_408 Y N Clostridium aerotolerans 546 X76163clade_408 Y N Clostridium aldenense 547 NR_043680 clade_408 Y NClostridium algidixylanolyticum 550 NR_028726 clade_408 Y N Clostridiumamygdalinum 552 AY353957 clade_408 Y N Clostridium asparagiforme 554ACCJ01000522 clade_408 Y N Clostridium bolteae 559 ABCC02000039clade_408 Y N Clostridium celerecrescens 566 JQ246092 clade_408 Y NClostridium citroniae 569 ADLJ01000059 clade_408 Y N Clostridiumclostridiiformes 571 M59089 clade_408 Y N Clostridium clostridioforme572 NR_044715 clade_408 Y N Clostridium hathewayi 590 AY552788 clade_408Y N Clostridium indolis 594 AF028351 clade_408 Y N Clostridium lavalense600 EF564277 clade_408 Y N Clostridium saccharolyticum 620 CP002109clade_408 Y N Clostridium sp. M62_1 633 ACFX02000046 clade_408 Y NClostridium sp. SS2_1 638 ABGC03000041 clade_408 Y N Clostridiumsphenoides 643 X73449 clade_408 Y N Clostridium symbiosum 652ADLQ01000114 clade_408 Y N Clostridium xylanolyticum 658 NR_037068clade_408 Y N Eubacterium hadrum 847 FR749933 clade_408 Y NLachnospiraceae bacterium 1052 ACTP01000124 clade_408 Y N 3_1_57FAA_CT1Lachnospiraceae bacterium 1055 ACTS01000081 clade_408 Y N 5_1_63FAALachnospiraceae bacterium A4 1059 DQ789118 clade_408 Y N Lachnospiraceaebacterium 1060 EU728771 clade_408 Y N DJF VP30 Lachnospiraceae genomosp. 1065 AY278618 clade_408 Y N C1 Clostridium difficile 578 NC_013315clade_409 Y OP Eubacterium sp. AS15b 862 HQ616364 clade_428 Y NEubacterium sp. OBRC9 863 HQ616354 clade_428 Y N Eubacterium sp. oralclone 871 AY947497 clade_428 Y N OH3A Eubacterium yurii 876 AEES01000073clade_428 Y N Clostridium acetobutylicum 545 NR_074511 clade_430 Y NClostridium algidicarnis 549 NR_041746 clade_430 Y N Clostridiumcadaveris 562 AB542932 clade_430 Y N Clostridium carboxidivorans 563FR733710 clade_430 Y N Clostridium estertheticum 580 NR_042153 clade_430Y N Clostridium fallax 581 NR_044714 clade_430 Y N Clostridium felsineum583 AF270502 clade_430 Y N Clostridium frigidicarnis 584 NR_024919clade_430 Y N Clostridium kluyveri 598 NR_074165 clade_430 Y NClostridium magnum 603 X77835 clade_430 Y N Clostridium putrefaciens 615NR_024995 clade_430 Y N Clostridium sp. HPB_46 629 AY862516 clade_430 YN Clostridium tyrobutyricum 656 NR_044718 clade_430 Y N Sutterellaparvirubra 1899 AB300989 clade_432 Y N Acetanaerobacterium 4 NR_042930clade_439 Y N elongatum Clostridium cellulosi 567 NR_044624 clade_439 YN Ethanoligenens harbinense 832 AY675965 clade_439 Y N Eubacteriumrectale 856 FP929042 clade_444 Y N Eubacterium sp. oral clone 865AY349374 clade_444 Y N GI038 Lachnobacterium bovis 1045 GU324407clade_444 Y N Roseburia cecicola 1634 GU233441 clade_444 Y N Roseburiafaecalis 1635 AY804149 clade_444 Y N Roseburia faecis 1636 AY305310clade_444 Y N Roseburia hominis 1637 AJ270482 clade_444 Y N Roseburiaintestinalis 1638 FP929050 clade_444 Y N Roseburia inulinivorans 1639AJ270473 clade_444 Y N Brevibacillus brevis 410 NR_041524 clade_448 Y NBrevibacillus laterosporus 414 NR_037005 clade_448 Y N Bacilluscoagulans 206 DQ297928 clade_451 Y OP Sporolactobacillus inulinus 1752NR_040962 clade_451 Y N Kocuria palustris 1041 EU333884 clade_453 Y NNocardia farcinica 1353 NC_006361 clade_455 Y N Bacillus sp. oral taxonF28 247 HM099650 clade_456 Y OP Catenibacterium mitsuokai 495 AB030224clade_469 Y N Clostridium sp. TM_40 640 AB249652 clade_469 Y NCoprobacillus cateniformis 670 AB030218 clade_469 Y N Coprobacillus sp.29_1 671 ADKX01000057 clade_469 Y N Clostridium rectum 618 NR_029271clade_470 Y N Eubacterium nodatum 854 U13041 clade_476 Y N Eubacteriumsaphenum 859 NR_026031 clade_476 Y N Eubacterium sp. oral clone 867AY349373 clade_476 Y N JH012 Eubacterium sp. oral clone 870 AY349378clade_476 Y N JS001 Faecalibacterium prausnitzii 880 ACOP02000011clade_478 Y N Gemmiger formicilis 932 GU562446 clade_478 Y NSubdoligranulum variabile 1896 AJ518869 clade_478 Y N Clostridiaceaebacterium JC13 532 JF824807 clade_479 Y N Clostridium sp. MLG055 634AF304435 clade_479 Y N Erysipelotrichaceae bacterium 822 ACTJ01000113clade_479 Y N 3_1_53 Clostridium cocleatum 575 NR_026495 clade_481 Y NClostridium ramosum 617 M23731 clade_481 Y N Clostridium saccharogumia619 DQ100445 clade_481 Y N Clostridium spiroforme 644 X73441 clade_481 YN Coprobacillus sp. D7 672 ACDT01000199 clade_481 Y N Clostridialesbacterium 535 AB477431 clade_482 Y N SY8519 Clostridium sp. SY8519 639AP012212 clade_482 Y N Eubacterium ramulus 855 AJ011522 clade_482 Y NErysipelothrix inopinata 819 NR_025594 clade_485 Y N Erysipelothrixrhusiopathiae 820 ACLK01000021 clade_485 Y N Erysipelothrix tonsillarum821 NR_040871 clade_485 Y N Holdemania filiformis 1004 Y11466 clade_485Y N Mollicutes bacterium pACH93 1258 AY297808 clade_485 Y N Coxiellaburnetii 736 CP000890 clade_486 Y Category-B Clostridium hiranonis 591AB023970 clade_487 Y N Clostridium irregulare 596 NR_029249 clade_487 YN Clostridium orbiscindens 609 Y18187 clade_494 Y N Clostridium sp. NML04A032 637 EU815224 clade_494 Y N Flavonifractor plautii 886 AY724678clade_494 Y N Pseudoflavonifractor capillosus 1591 AY136666 clade_494 YN Ruminococcaceae bacterium 1655 ADDX01000083 clade_494 Y N D16Acetivibrio cellulolyticus 5 NR_025917 clade_495 Y N Clostridiumaldrichii 548 NR_026099 clade_495 Y N Clostridium clariflavum 570NR_041235 clade_495 Y N Clostridium stercorarium 647 NR_025100 clade_495Y N Clostridium straminisolvens 649 NR_024829 clade_495 Y N Clostridiumthermocellum 655 NR_074629 clade_495 Y N Fusobacterium nucleatum 901ADVK01000034 clade_497 Y N Eubacterium barkeri 834 NR_044661 clade_512 YN Eubacterium callanderi 838 NR_026330 clade_512 Y N Eubacterium limosum850 CP002273 clade_512 Y N Anaerotruncus colihominis 164 ABGD02000021clade_516 Y N Clostridium methylpentosum 606 ACEC01000059 clade_516 Y NClostridium sp. YIT 12070 642 AB491208 clade_516 Y NHydrogenoanaerobacterium 1005 NR_044425 clade_516 Y N saccharovoransRuminococcus albus 1656 AY445600 clade_516 Y N Ruminococcus flavefaciens1660 NR_025931 clade_516 Y N Clostridium haemolyticum 589 NR_024749clade_517 Y N Clostridium novyi 608 NR_074343 clade_517 Y N Clostridiumsp. LMG 16094 632 X95274 clade_517 Y N Eubacterium ventriosum 874 L34421clade_519 Y N Bacteroides galacturonicus 280 DQ497994 clade_522 Y NEubacterium eligens 845 CP001104 clade_522 Y N Lachnospira multipara1046 FR733699 clade_522 Y N Lachnospira pectinoschiza 1047 L14675clade_522 Y N Lactobacillus rogosae 1114 GU269544 clade_522 Y N Bacillushorti 214 NR_036860 clade_527 Y OP Bacillus sp. 9_3AIA 232 FN397519clade_527 Y OP Eubacterium brachy 836 U13038 clade_533 Y N Filifactoralocis 881 CP002390 clade_533 Y N Filifactor villosus 882 NR_041928clade_533 Y N Clostridium leptum 601 AJ305238 clade_537 Y N Clostridiumsp. YIT 12069 641 AB491207 clade_537 Y N Clostridium sporosphaeroides646 NR_044835 clade_537 Y N Eubacterium 841 HM037995 clade_537 Y Ncoprostanoligenes Ruminococcus bromii 1657 EU266549 clade_537 Y NEubacterium siraeum 860 ABCA03000054 clade_538 Y N Clostridium viride657 NR_026204 clade_540 Y N Oscillibacter sp. G2 1386 HM626173 clade_540Y N Oscillibacter valericigenes 1387 NR_074793 clade_540 Y NOscillospira guilliermondii 1388 AB040495 clade_540 Y N Butyrivibriocrossotus 455 ABWN01000012 clade_543 Y N Clostridium sp. L2_50 631AAYW02000018 clade_543 Y N Coprococcus eutactus 675 EF031543 clade_543 YN Coprococcus sp. ART55_1 676 AY350746 clade_543 Y N Eubacteriumruminantium 857 NR_024661 clade_543 Y N Collinsella aerofaciens 659AAVN02000007 clade_553 Y N Alkaliphilus metalliredigenes 137 AY137848clade_554 Y N Alkaliphilus oremlandii 138 NR_043674 clade_554 Y NClostridium sticklandii 648 L04167 clade_554 Y N Turicibacter sanguinis1965 AF349724 clade_555 Y N Fulvimonas sp. NML 060897 892 EF589680clade_557 Y N Desulfitobacterium frappieri 753 AJ276701 clade_560 Y NDesulfitobacterium hafniense 754 NR_074996 clade_560 Y NDesulfotomaculum nigrificans 756 NR_044832 clade_560 Y N Lutisporathermophila 1191 NR_041236 clade_564 Y N Brachyspira pilosicoli 405NR_075069 clade_565 Y N Eggerthella lenta 778 AF292375 clade_566 Y NStreptomyces albus 1888 AJ697941 clade_566 Y N Chlamydiales bacteriumNS11 505 JN606074 clade_567 Y N Anaerofustis stercorihominis 159ABIL02000005 clade_570 Y N Butyricicoccus pullicaecorum 453 HH793440clade_572 Y N Eubacterium desmolans 843 NR_044644 clade_572 Y NPapillibacter cinnamivorans 1415 NR_025025 clade_572 Y N Sporobactertermitidis 1751 NR_044972 clade_572 Y N Deferribacteres sp. oral clone744 AY349371 clade_575 Y N JV006 Clostridium colinum 577 NR_026151clade_576 Y N Clostridium lactatifermentans 599 NR_025651 clade_576 Y NClostridium piliforme 614 D14639 clade_576 Y N Saccharomonospora viridis1671 X54286 clade_579 Y N Thermobifida fusca 1921 NC_007333 clade_579 YN Leptospira licerasiae 1164 EF612284 clade_585 Y OP Moorellathermoacetica 1259 NR_075001 clade_590 Y N Thermoanaerobacter 1920CP000924 clade_590 Y N pseudethanolicus Flexistipes sinusarabici 888NR_074881 clade_591 Y N Gloeobacter violaceus 942 NR_074282 clade_596 YN Eubacterium sp. oral clone 869 AY349377 clade_90 Y N JN088 Clostridiumoroticum 610 FR749922 clade_96 Y N Clostridium sp. D5 627 ADBG01000142clade_96 Y N Eubacterium contortum 840 FR749946 clade_96 Y N Eubacteriumfissicatena 846 FR749935 clade_96 Y N Corynebacterium coyleae 692 X96497clade_100 N N Corynebacterium mucifaciens 711 NR_026396 clade_100 N NCorynebacterium 733 AM397636 clade_100 N N ureicelerivoransCorynebacterium appendicis 684 NR_028951 clade_102 N N Corynebacteriumgenitalium 698 ACLJ01000031 clade_102 N N Corynebacterium glaucum 699NR_028971 clade_102 N N Corynebacterium imitans 703 AF537597 clade_102 NN Corynebacterium riegelii 719 EU848548 clade_102 N N Corynebacteriumsp. 723 HE575405 clade_102 N N L_2012475 Corynebacterium sp. NML 724GU238409 clade_102 N N 93_0481 Corynebacterium 728 Y09655 clade_102 N Nsundsvallense Corynebacterium tuscaniae 730 AY677186 clade_102 N NPrevotella maculosa 1504 AGEK01000035 clade_104 N N Prevotella oris 1513ADDV01000091 clade_104 N N Prevotella salivae 1517 AB108826 clade_104 NN Prevotella sp. ICM55 1521 HQ616399 clade_104 N N Prevotella sp. oralclone 1528 AY005057 clade_104 N N AA020 Prevotella sp. oral clone GI0321538 AY349396 clade_104 N N Prevotella sp. oral taxon G70 1558 GU432179clade_104 N N Prevotella corporis 1491 L16465 clade_105 N N Bacteroidessp. 4_1_36 312 ACTC01000133 clade_110 N N Bacteroides sp. AR20 315AF139524 clade_110 N N Bacteroides sp. D20 319 ACPT01000052 clade_110 NN Bacteroides sp. F_4 322 AB470322 clade_110 N N Bacteroides uniformis329 AB050110 clade_110 N N Prevotella nanceiensis 1510 JN867228clade_127 N N Prevotella sp. oral taxon 299 1548 ACWZ01000026 clade_127N N Prevotella bergensis 1485 ACKS01000100 clade_128 N N Prevotellabuccalis 1489 JN867261 clade_129 N N Prevotella timonensis 1564ADEF01000012 clade_129 N N Prevotella oralis 1512 AEPE01000021 clade_130N N Prevotella sp. SEQ072 1525 JN867238 clade_130 N N Leuconostoccarnosum 1177 NR_040811 clade_135 N N Leuconostoc gasicomitatum 1179FN822744 clade_135 N N Leuconostoc inhae 1180 NR_025204 clade_135 N NLeuconostoc kimchii 1181 NR_075014 clade_135 N N Edwardsiella tarda 777CP002154 clade_139 N N Photorhabdus asymbiotica 1466 Z76752 clade_139 NN Psychrobacter arcticus 1607 CP000082 clade_141 N N Psychrobactercibarius 1608 HQ698586 clade_141 N N Psychrobacter cryohalolentis 1609CP000323 clade_141 N N Psychrobacter faecalis 1610 HQ698566 clade_141 NN Psychrobacter nivimaris 1611 HQ698587 clade_141 N N Psychrobacterpulmonis 1612 HQ698582 clade_141 N N Pseudomonas aeruginosa 1592AABQ07000001 clade_154 N N Pseudomonas sp. 2_1_26 1600 ACWU01000257clade_154 N N Corynebacterium confusum 691 Y15886 clade_158 N NCorynebacterium propinquum 712 NR_037038 clade_158 N N Corynebacterium713 X84258 clade_158 N N pseudodiphtheriticum Bartonella bacilliformis338 NC_008783 clade_159 N N Bartonella grahamii 339 CP001562 clade_159 NN Bartonella henselae 340 NC_005956 clade_159 N N Bartonella quintana341 BX897700 clade_159 N N Bartonella tamiae 342 EF672728 clade_159 N NBartonella washoensis 343 FJ719017 clade_159 N N Brucella abortus 430ACBJ01000075 clade_159 N Category-B Brucella canis 431 NR_044652clade_159 N Category-B Brucella ceti 432 ACJD01000006 clade_159 NCategory-B Brucella melitensis 433 AE009462 clade_159 N Category-BBrucella microti 434 NR_042549 clade_159 N Category-B Brucella ovis 435NC_009504 clade_159 N Category-B Brucella sp. 83_13 436 ACBQ01000040clade_159 N Category-B Brucella sp. BO1 437 EU053207 clade_159 NCategory-B Brucella suis 438 ACBK01000034 clade_159 N Category-BOchrobactrum anthropi 1360 NC_009667 clade_159 N N Ochrobactrumintermedium 1361 ACQA01000001 clade_159 N N Ochrobactrum 1362 DQ365921clade_159 N N pseudintermedium Prevotella genomo sp. C2 1496 AY278625clade_164 N N Prevotella multisaccharivorax 1509 AFJE01000016 clade_164N N Prevotella sp. oral clone 1543 AY550997 clade_164 N N IDR_CEC_0055Prevotella sp. oral taxon 292 1547 GQ422735 clade_164 N N Prevotella sp.oral taxon 300 1549 GU409549 clade_164 N N Prevotella marshii 1505AEEI01000070 clade_166 N N Prevotella sp. oral clone IK053 1544 AY349401clade_166 N N Prevotella sp. oral taxon 781 1554 GQ422744 clade_166 N NPrevotella stercorea 1562 AB244774 clade_166 N N Prevotella brevis 1487NR_041954 clade_167 N N Prevotella ruminicola 1516 CP002006 clade_167 NN Prevotella sp. sp24 1560 AB003384 clade_167 N N Prevotella sp. sp341561 AB003385 clade_167 N N Prevotella albensis 1483 NR_025300 clade_168N N Prevotella copri 1490 ACBX02000014 clade_168 N N Prevotella oulorum1514 L16472 clade_168 N N Prevotella sp. BI_42 1518 AJ581354 clade_168 NN Prevotella sp. oral clone 1546 AY207050 clade_168 N N P4PB_83 P2Prevotella sp. oral taxon G60 1557 GU432133 clade_168 N N Prevotellaamnii 1484 AB547670 clade_169 N N Bacteroides caccae 268 EU136686clade_170 N N Bacteroides finegoldii 277 AB222699 clade_170 N NBacteroides intestinalis 283 ABJL02000006 clade_171 N N Bacteroides sp.XB44A 326 AM230649 clade_171 N N Bifidobacteriaceae genomo sp. 345AY278612 clade_172 N N C1 Bifidobacterium adolescentis 346 AAXD02000018clade_172 N N Bifidobacterium angulatum 347 ABYS02000004 clade_172 N NBifidobacterium animalis 348 CP001606 clade_172 N N Bifidobacteriumbreve 350 CP002743 clade_172 N N Bifidobacterium catenulatum 351ABXY01000019 clade_172 N N Bifidobacterium dentium 352 CP001750clade_172 N OP Bifidobacterium gallicum 353 ABXB03000004 clade_172 N NBifidobacterium infantis 354 AY151398 clade_172 N N Bifidobacterium 355AB491757 clade_172 N N kashiwanohense Bifidobacterium longum 356ABQQ01000041 clade_172 N N Bifidobacterium 357 ABXX02000002 clade_172 NN pseudocatenulatum Bifidobacterium pseudolongum 358 NR_043442 clade_172N N Bifidobacterium scardovii 359 AJ307005 clade_172 N N Bifidobacteriumsp. HM2 360 AB425276 clade_172 N N Bifidobacterium sp. HMLN12 361JF519685 clade_172 N N Bifidobacterium sp. M45 362 HM626176 clade_172 NN Bifidobacterium sp. MSX5B 363 HQ616382 clade_172 N N Bifidobacteriumsp. TM_7 364 AB218972 clade_172 N N Bifidobacterium thermophilum 365DQ340557 clade_172 N N Leuconostoc citreum 1178 AM157444 clade_175 N NLeuconostoc lactis 1182 NR_040823 clade_175 N N Alicyclobacillusacidoterrestris 123 NR_040844 clade_179 N N Alicyclobacillus 125NR_024754 clade_179 N N cycloheptanicus Acinetobacter baumannii 27ACYQ01000014 clade_181 N N Acinetobacter calcoaceticus 28 AM157426clade_181 N N Acinetobacter genomo sp. C1 29 AY278636 clade_181 N NAcinetobacter haemolyticus 30 ADMT01000017 clade_181 N N Acinetobacterjohnsonii 31 ACPL01000162 clade_181 N N Acinetobacter junii 32ACPM01000135 clade_181 N N Acinetobacter lwoffii 33 ACPN01000204clade_181 N N Acinetobacter parvus 34 AIEB01000124 clade_181 N NAcinetobacter schindleri 36 NR_025412 clade_181 N N Acinetobacter sp.56A1 37 GQ178049 clade_181 N N Acinetobacter sp. CIP 101934 38 JQ638573clade_181 N N Acinetobacter sp. CIP 102143 39 JQ638578 clade_181 N NAcinetobacter sp. M16_22 41 HM366447 clade_181 N N Acinetobacter sp.RUH2624 42 ACQF01000094 clade_181 N N Acinetobacter sp. SH024 43ADCH01000068 clade_181 N N Lactobacillus jensenii 1092 ACQD01000066clade_182 N N Alcaligenes faecalis 119 AB680368 clade_183 N NAlcaligenes sp. CO14 120 DQ643040 clade_183 N N Alcaligenes sp. S3 121HQ262549 clade_183 N N Oligella ureolytica 1366 NR_041998 clade_183 N NOligella urethralis 1367 NR_041753 clade_183 N N Eikenella corrodens 784ACEA01000028 clade_185 N N Kingella denitrificans 1019 AEWV01000047clade_185 N N Kingella genomo sp. P1 oral 1020 DQ003616 clade_185 N Ncone MB2_C20 Kingella kingae 1021 AFHS01000073 clade_185 N N Kingellaoralis 1022 ACJW02000005 clade_185 N N Kingella sp. oral clone ID0591023 AY349381 clade_185 N N Neisseria elongata 1330 ADBF01000003clade_185 N N Neisseria genomo sp. P2 oral 1332 DQ003630 clade_185 N Nclone MB5_P15 Neisseria sp. oral clone JC012 1345 AY349388 clade_185 N NNeisseria sp. SMC_A9199 1342 FJ763637 clade_185 N N Simonsiella muelleri1731 ADCY01000105 clade_185 N N Corynebacterium 700 ABYP01000081clade_193 N N glucuronolyticum Corynebacterium 716 FJ185225 clade_193 NN pyruviciproducens Rothia aeria 1649 DQ673320 clade_194 N N Rothiadentocariosa 1650 ADDW01000024 clade_194 N N Rothia sp. oral taxon 1881653 GU470892 clade_194 N N Corynebacterium accolens 681 ACGD01000048clade_195 N N Corynebacterium macginleyi 707 AB359393 clade_195 N NCorynebacterium 714 ABYQ01000237 clade_195 N N pseudogenitaliumCorynebacterium 729 ACVP01000009 clade_195 N N tuberculostearicumLactobacillus casei 1074 CP000423 clade_198 N N Lactobacillus paracasei1106 ABQV01000067 clade_198 N N Lactobacillus zeae 1143 NR_037122clade_198 N N Prevotella dentalis 1492 AB547678 clade_205 N N Prevotellasp. oral clone 1529 AY923148 clade_206 N N ASCG10 Prevotella sp. oralclone 1541 AY349399 clade_206 N N HF050 Prevotella sp. oral clone ID0191542 AY349400 clade_206 N N Prevotella sp. oral clone IK062 1545AY349402 clade_206 N N Prevotella genomo sp. P9 oral 1499 DQ003633clade_207 N N clone MB7_G16 Prevotella sp. oral clone 1531 AY005062clade_207 N N AU069 Prevotella sp. oral clone 1532 AY005063 clade_207 NN CY006 Prevotella sp. oral clone FL019 1534 AY349392 clade_207 N NActinomyces genomo sp. C1 56 AY278610 clade_212 N N Actinomyces genomosp. C2 57 AY278611 clade_212 N N Actinomyces genomo sp. P1 58 DQ003632clade_212 N N oral clone MB6_C03 Actinomyces georgiae 59 GU561319clade_212 N N Actinomyces israelii 60 AF479270 clade_212 N N Actinomycesmassiliensis 61 AB545934 clade_212 N N Actinomyces meyeri 62 GU561321clade_212 N N Actinomyces odontolyticus 66 ACYT01000123 clade_212 N NActinomyces orihominis 68 AJ575186 clade_212 N N Actinomyces sp. CCUG37290 71 AJ234058 clade_212 N N Actinomyces sp. ICM34 75 HQ616391clade_212 N N Actinomyces sp. ICM41 76 HQ616392 clade_212 N NActinomyces sp. ICM47 77 HQ616395 clade_212 N N Actinomyces sp. ICM54 78HQ616398 clade_212 N N Actinomyces sp. oral clone 87 AY349366 clade_212N N IP081 Actinomyces sp. oral taxon 91 AEUH01000060 clade_212 N N 178Actinomyces sp. oral taxon 92 AEPP01000041 clade_212 N N 180 Actinomycessp. TeJ5 80 GU561315 clade_212 N N Haematobacter sp. BC14248 968GU396991 clade_213 N N Paracoccus denitrificans 1424 CP000490 clade_213N N Paracoccus marcusii 1425 NR_044922 clade_213 N N Grimontia hollisae967 ADAQ01000013 clade_216 N N Shewanella putrefaciens 1723 CP002457clade_216 N N Afipia genomo sp. 4 111 EU117385 clade_217 N NRhodopseudomonas palustris 1626 CP000301 clade_217 N N Methylobacteriumextorquens 1223 NC_010172 clade_218 N N Methylobacterium podarium 1224AY468363 clade_218 N N Methylobacterium 1225 GU294320 clade_218 N Nradiotolerans Methylobacterium sp. 1sub 1226 AY468371 clade_218 N NMethylobacterium sp. MM4 1227 AY468370 clade_218 N N Achromobacterdenitrificans 18 NR_042021 clade_224 N N Achromobacter piechaudii 19ADMS01000149 clade_224 N N Achromobacter xylosoxidans 20 ACRC01000072clade_224 N N Bordetella bronchiseptica 384 NR_025949 clade_224 N OPBordetella holmesii 385 AB683187 clade_224 N OP Bordetella parapertussis386 NR_025950 clade_224 N OP Bordetella pertussis 387 BX640418 clade_224N OP Microbacterium chocolatum 1230 NR_037045 clade_225 N NMicrobacterium flavescens 1231 EU714363 clade_225 N N Microbacteriumlacticum 1233 EU714351 clade_225 N N Microbacterium oleivorans 1234EU714381 clade_225 N N Microbacterium oxydans 1235 EU714348 clade_225 NN Microbacterium paraoxydans 1236 AJ491806 clade_225 N N Microbacterium1237 EU714359 clade_225 N N phyllosphaerae Microbacterium schleiferi1238 NR_044936 clade_225 N N Microbacterium sp. 768 1239 EU714378clade_225 N N Microbacterium sp. oral strain 1240 AF287752 clade_225 N NC24KA Microbacterium testaceum 1241 EU714365 clade_225 N NCorynebacterium atypicum 686 NR_025540 clade_229 N N Corynebacteriummastitidis 708 AB359395 clade_229 N N Corynebacterium sp. NML 725GU238411 clade_229 N N 97_0186 Mycobacterium elephantis 1275 AF385898clade_237 N OP Mycobacterium paraterrae 1288 EU919229 clade_237 N OPMycobacterium phlei 1289 GU142920 clade_237 N OP Mycobacterium sp. 17761293 EU703152 clade_237 N N Mycobacterium sp. 1781 1294 EU703147clade_237 N N Mycobacterium sp. AQ1GA4 1297 HM210417 clade_237 N NMycobacterium sp. GN_10546 1299 FJ497243 clade_237 N N Mycobacterium sp.GN_10827 1300 FJ497247 clade_237 N N Mycobacterium sp. GN_11124 1301FJ652846 clade_237 N N Mycobacterium sp. GN_9188 1302 FJ497240 clade_237N N Mycobacterium sp. 1303 FJ555538 clade_237 N N GR_2007_210Anoxybacillus contaminans 172 NR_029006 clade_238 N N Bacillus aeolius195 NR_025557 clade_238 N N Brevibacterium frigoritolerans 422 NR_042639clade_238 N N Geobacillus sp. E263 934 DQ647387 clade_238 N NGeobacillus sp. WCH70 935 CP001638 clade_238 N N Geobacillusthermocatenulatus 937 NR_043020 clade_238 N N Geobacillusthermoleovorans 940 NR_074931 clade_238 N N Lysinibacillus fusiformis1192 FN397522 clade_238 N N Planomicrobium koreense 1468 NR_025011clade_238 N N Sporosarcina newyorkensis 1754 AFPZ01000142 clade_238 N NSporosarcina sp. 2681 1755 GU994081 clade_238 N N Ureibacillus composti1968 NR_043746 clade_238 N N Ureibacillus suwonensis 1969 NR_043232clade_238 N N Ureibacillus terrenus 1970 NR_025394 clade_238 N NUreibacillus thermophilus 1971 NR_043747 clade_238 N N Ureibacillusthermosphaericus 1972 NR_040961 clade_238 N N Prevotella micans 1507AGWK01000061 clade_239 N N Prevotella sp. oral clone 1533 AY005065clade_239 N N DA058 Prevotella sp. SEQ053 1523 JN867222 clade_239 N NTreponema socranskii 1937 NR_024868 clade_240 N OP Treponema sp.6:H:D15A_4 1938 AY005083 clade_240 N N Treponema sp. oral taxon 265 1953GU408850 clade_240 N N Treponema sp. oral taxon G85 1958 GU432215clade_240 N N Porphyromonas endodontalis 1472 ACNN01000021 clade_241 N NPorphyromonas sp. oral clone 1478 AY005068 clade_241 N N BB134Porphyromonas sp. oral clone 1479 AY005069 clade_241 N N F016Porphyromonas sp. oral clone 1480 AY207054 clade_241 N N P2PB_52 P1Porphyromonas sp. oral clone 1481 AY207057 clade_241 N N P4GB_100 P2Acidovorax sp. 98_63833 26 AY258065 clade_245 N N Comamonadaceaebacterium 663 JN585335 clade_245 N N NML000135 Comamonadaceae bacterium664 JN585331 clade_245 N N NML790751 Comamonadaceae bacterium 665JN585332 clade_245 N N NML910035 Comamonadaceae bacterium 666 JN585333clade_245 N N NML910036 Comamonas sp. NSP5 668 AB076850 clade_245 N NDelftia acidovorans 748 CP000884 clade_245 N N Xenophilus aerolatus 2018JN585329 clade_245 N N Oribacterium sp. oral taxon 1380 ACIQ02000009clade_246 N N 078 Oribacterium sp. oral taxon 1381 GQ422713 clade_246 NN 102 Weissella cibaria 2007 NR_036924 clade_247 N N Weissella confusa2008 NR_040816 clade_247 N N Weissella hellenica 2009 AB680902 clade_247N N Weissella kandleri 2010 NR_044659 clade_247 N N Weissella koreensis2011 NR_075058 clade_247 N N Weissella paramesenteroides 2012ACKU01000017 clade_247 N N Weissella sp. KLDS 7.0701 2013 EU600924clade_247 N N Mobiluncus curtisii 1251 AEPZ01000013 clade_249 N NEnhydrobacter aerosaccus 785 ACYI01000081 clade_256 N N Moraxellaosloensis 1262 JN175341 clade_256 N N Moraxella sp. GM2 1264 JF837191clade_256 N N Brevibacterium casei 420 JF951998 clade_257 N NBrevibacterium epidermidis 421 NR_029262 clade_257 N N Brevibacteriumsanguinis 426 NR_028016 clade_257 N N Brevibacterium sp. H15 427AB177640 clade_257 N N Acinetobacter radioresistens 35 ACVR01000010clade_261 N N Lactobacillus alimentarius 1068 NR_044701 clade_263 N NLactobacillus farciminis 1082 NR_044707 clade_263 N N Lactobacilluskimchii 1097 NR_025045 clade_263 N N Lactobacillus nodensis 1101NR_041629 clade_263 N N Lactobacillus tucceti 1138 NR_042194 clade_263 NN Pseudomonas mendocina 1595 AAUL01000021 clade_265 N N Pseudomonas 1598NR_037000 clade_265 N N pseudoalcaligenes Pseudomonas sp. NP522b 1602EU723211 clade_265 N N Pseudomonas stutzeri 1603 AM905854 clade_265 N NPaenibacillus barcinonensis 1390 NR_042272 clade_270 N N Paenibacillusbarengoltzii 1391 NR_042756 clade_270 N N Paenibacillus chibensis 1392NR_040885 clade_270 N N Paenibacillus cookii 1393 NR_025372 clade_270 NN Paenibacillus durus 1394 NR_037017 clade_270 N N Paenibacillusglucanolyticus 1395 D78470 clade_270 N N Paenibacillus lactis 1396NR_025739 clade_270 N N Paenibacillus pabuli 1398 NR_040853 clade_270 NN Paenibacillus popilliae 1400 NR_040888 clade_270 N N Paenibacillus sp.CIP 101062 1401 HM212646 clade_270 N N Paenibacillus sp. JC66 1404JF824808 clade_270 N N Paenibacillus sp. R_27413 1405 HE586333 clade_270N N Paenibacillus sp. R_27422 1406 HE586338 clade_270 N N Paenibacillustimonensis 1408 NR_042844 clade_270 N N Rothia mucilaginosa 1651ACVO01000020 clade_271 N N Rothia nasimurium 1652 NR_025310 clade_271 NN Prevotella sp. oral taxon 302 1550 ACZK01000043 clade_280 N NPrevotella sp. oral taxon F68 1556 HM099652 clade_280 N N Prevotellatannerae 1563 ACIJ02000018 clade_280 N N Prevotellaceae bacterium 1566AY207061 clade_280 N N P4P_62 P1 Porphyromonas 1471 AENO01000048clade_281 N N asaccharolytica Porphyromonas gingivalis 1473 AE015924clade_281 N N Porphyromonas macacae 1475 NR_025908 clade_281 N NPorphyromonas sp. UQD 301 1477 EU012301 clade_281 N N Porphyromonasuenonis 1482 ACLR01000152 clade_281 N N Leptotrichia buccalis 1165CP001685 clade_282 N N Leptotrichia hofstadii 1168 ACVB02000032clade_282 N N Leptotrichia sp. oral clone 1173 AY349386 clade_282 N NHE012 Leptotrichia sp. oral taxon 223 1176 GU408547 clade_282 N NBacteroides fluxus 278 AFBN01000029 clade_285 N N Bacteroides helcogenes281 CP002352 clade_285 N N Parabacteroides johnsonii 1419 ABYH01000014clade_286 N N Parabacteroides merdae 1420 EU136685 clade_286 N NTreponema denticola 1926 ADEC01000002 clade_288 N OP Treponema genomosp. P5 oral 1929 DQ003624 clade_288 N N clone MB3_P23 Treponema putidum1935 AJ543428 clade_288 N OP Treponema sp. oral clone 1942 AY207055clade_288 N N P2PB_53 P3 Treponema sp. oral taxon 247 1949 GU408748clade_288 N N Treponema sp. oral taxon 250 1950 GU408776 clade_288 N NTreponema sp. oral taxon 251 1951 GU408781 clade_288 N N Anaerococcushydrogenalis 144 ABXA01000039 clade_289 N N Anaerococcus sp. 8404299 148HM587318 clade_289 N N Anaerococcus sp. gpac215 156 AM176540 clade_289 NN Anaerococcus vaginalis 158 ACXU01000016 clade_289 N NPropionibacterium 1569 NC_019395 clade_290 N N acidipropioniciPropionibacterium avidum 1571 AJ003055 clade_290 N N Propionibacteriumgranulosum 1573 FJ785716 clade_290 N N Propionibacterium jensenii 1574NR_042269 clade_290 N N Propionibacterium propionicum 1575 NR_025277clade_290 N N Propionibacterium sp. H456 1577 AB177643 clade_290 N NPropionibacterium thoenii 1581 NR_042270 clade_290 N N Bifidobacteriumbifidum 349 ABQP01000027 clade_293 N N Leuconostoc mesenteroides 1183ACKV01000113 clade_295 N N Leuconostoc 1184 NR_040814 clade_295 N Npseudomesenteroides Johnsonella ignava 1016 X87152 clade_298 N NPropionibacterium acnes 1570 ADJM01000010 clade_299 N NPropionibacterium sp. 1576 AFIL01000035 clade_299 N N 434_HC2Propionibacterium sp. LG 1578 AY354921 clade_299 N N Propionibacteriumsp. S555a 1579 AB264622 clade_299 N N Alicyclobacillus sp. CCUG 128HE613268 clade_301 N N 53762 Actinomyces cardiffensis 53 GU470888clade_303 N N Actinomyces funkei 55 HQ906497 clade_303 N N Actinomycessp. HKU31 74 HQ335393 clade_303 N N Actinomyces sp. oral taxon 94HM099646 clade_303 N N C55 Kerstersia gyiorum 1018 NR_025669 clade_307 NN Pigmentiphaga daeguensis 1467 JN585327 clade_307 N N Aeromonasallosaccharophila 104 S39232 clade_308 N N Aeromonas enteropelogenes 105X71121 clade_308 N N Aeromonas hydrophila 106 NC_008570 clade_308 N NAeromonas jandaei 107 X60413 clade_308 N N Aeromonas salmonicida 108NC_009348 clade_308 N N Aeromonas trota 109 X60415 clade_308 N NAeromonas veronii 110 NR_044845 clade_308 N N Marvinbryantiaformatexigens 1196 AJ505973 clade_309 N N Rhodobacter sp. oral taxon1620 HM099648 clade_310 N N C30 Rhodobacter sphaeroides 1621 CP000144clade_310 N N Lactobacillus antri 1071 ACLL01000037 clade_313 N NLactobacillus coleohominis 1076 ACOH01000030 clade_313 N N Lactobacillusfermentum 1083 CP002033 clade_313 N N Lactobacillus gastricus 1085AICN01000060 clade_313 N N Lactobacillus mucosae 1099 FR693800 clade_313N N Lactobacillus oris 1103 AEKL01000077 clade_313 N N Lactobacilluspontis 1111 HM218420 clade_313 N N Lactobacillus reuteri 1112ACGW02000012 clade_313 N N Lactobacillus sp. KLDS 1.0707 1127 EU600911clade_313 N N Lactobacillus sp. KLDS 1.0709 1128 EU600913 clade_313 N NLactobacillus sp. KLDS 1.0711 1129 EU600915 clade_313 N N Lactobacillussp. KLDS 1.0713 1131 EU600917 clade_313 N N Lactobacillus sp. KLDS1.0716 1132 EU600921 clade_313 N N Lactobacillus sp. KLDS 1.0718 1133EU600922 clade_313 N N Lactobacillus sp. oral taxon 1137 GQ422710clade_313 N N 052 Lactobacillus vaginalis 1140 ACGV01000168 clade_313 NN Brevibacterium aurantiacum 419 NR_044854 clade_314 N N Brevibacteriumlinens 423 AJ315491 clade_314 N N Lactobacillus pentosus 1108 JN813103clade_315 N N Lactobacillus plantarum 1110 ACGZ02000033 clade_315 N NLactobacillus sp. KLDS 1.0702 1123 EU600906 clade_315 N N Lactobacillussp. KLDS 1.0703 1124 EU600907 clade_315 N N Lactobacillus sp. KLDS1.0704 1125 EU600908 clade_315 N N Lactobacillus sp. KLDS 1.0705 1126EU600909 clade_315 N N Agrobacterium radiobacter 115 CP000628 clade_316N N Agrobacterium tumefaciens 116 AJ389893 clade_316 N N Corynebacterium685 EF463055 clade_317 N N argentoratense Corynebacterium diphtheriae693 NC_002935 clade_317 N OP Corynebacterium 715 NR_037070 clade_317 N Npseudotuberculosis Corynebacterium renale 717 NR_037069 clade_317 N NCorynebacterium ulcerans 731 NR_074467 clade_317 N N Aurantimonascoralicida 191 AY065627 clade_318 N N Aureimonas altamirensis 192FN658986 clade_318 N N Lactobacillus acidipiscis 1066 NR_024718clade_320 N N Lactobacillus salivarius 1117 AEBA01000145 clade_320 N NLactobacillus sp. KLDS 1.0719 1134 EU600923 clade_320 N N Lactobacillusbuchneri 1073 ACGH01000101 clade_321 N N Lactobacillus genomo sp. C11086 AY278619 clade_321 N N Lactobacillus genomo sp. C2 1087 AY278620clade_321 N N Lactobacillus hilgardii 1089 ACGP01000200 clade_321 N NLactobacillus kefiri 1096 NR_042230 clade_321 N N Lactobacillusparabuchneri 1105 NR_041294 clade_321 N N Lactobacillus parakefiri 1107NR_029039 clade_321 N N Lactobacillus curvatus 1079 NR_042437 clade_322N N Lactobacillus sakei 1116 DQ989236 clade_322 N N Aneurinibacillusaneurinilyticus 167 AB101592 clade_323 N N Aneurinibacillus danicus 168NR_028657 clade_323 N N Aneurinibacillus migulanus 169 NR_036799clade_323 N N Aneurinibacillus terranovensis 170 NR_042271 clade_323 N NStaphylococcus aureus 1757 CP002643 clade_325 N Category-BStaphylococcus auricularis 1758 JQ624774 clade_325 N N Staphylococcuscapitis 1759 ACFR01000029 clade_325 N N Staphylococcus caprae 1760ACRH01000033 clade_325 N N Staphylococcus carnosus 1761 NR_075003clade_325 N N Staphylococcus cohnii 1762 JN175375 clade_325 N NStaphylococcus condimenti 1763 NR_029345 clade_325 N N Staphylococcusepidermidis 1764 ACHE01000056 clade_325 N N Staphylococcus equorum 1765NR_027520 clade_325 N N Staphylococcus haemolyticus 1767 NC_007168clade_325 N N Staphylococcus hominis 1768 AM157418 clade_325 N NStaphylococcus lugdunensis 1769 AEQA01000024 clade_325 N NStaphylococcus pasteuri 1770 FJ189773 clade_325 N N Staphylococcus 1771CP002439 clade_325 N N pseudintermedius Staphylococcus 1772 NR_029158clade_325 N N saccharolyticus Staphylococcus saprophyticus 1773NC_007350 clade_325 N N Staphylococcus sp. clone 1777 AF467424 clade_325N N bottae7 Staphylococcus sp. H292 1775 AB177642 clade_325 N NStaphylococcus sp. H780 1776 AB177644 clade_325 N N Staphylococcussuccinus 1778 NR_028667 clade_325 N N Staphylococcus warneri 1780ACPZ01000009 clade_325 N N Staphylococcus xylosus 1781 AY395016clade_325 N N Cardiobacterium hominis 490 ACKY01000036 clade_326 N NCardiobacterium valvarum 491 NR_028847 clade_326 N N Pseudomonasfluorescens 1593 AY622220 clade_326 N N Pseudomonas gessardii 1594FJ943496 clade_326 N N Pseudomonas monteilii 1596 NR_024910 clade_326 NN Pseudomonas poae 1597 GU188951 clade_326 N N Pseudomonas putida 1599AF094741 clade_326 N N Pseudomonas sp. G1229 1601 DQ910482 clade_326 N NPseudomonas tolaasii 1604 AF320988 clade_326 N N Pseudomonas viridiflava1605 NR_042764 clade_326 N N Listeria grayi 1185 ACCR02000003 clade_328N OP Listeria innocua 1186 JF967625 clade_328 N N Listeria ivanovii 1187X56151 clade_328 N N Listeria monocytogenes 1188 CP002003 clade_328 NCategory-B Listeria welshimeri 1189 AM263198 clade_328 N OPCapnocytophaga sp. oral clone 484 AY923149 clade_333 N N ASCH05Capnocytophaga sputigena 489 ABZV01000054 clade_333 N N Leptotrichiagenomo sp. C1 1166 AY278621 clade_334 N N Leptotrichia shahii 1169AY029806 clade_334 N N Leptotrichia sp. 1170 AF189244 clade_334 N Nneutropenic Patient Leptotrichia sp. oral clone 1171 AY349384 clade_334N N GT018 Leptotrichia sp. oral clone 1172 AY349385 clade_334 N N GT020Bacteroides sp. 20_3 296 ACRQ01000064 clade_335 N N Bacteroides sp.3_1_19 307 ADCJ01000062 clade_335 N N Bacteroides sp. 3_2_5 311ACIB01000079 clade_335 N N Parabacteroides distasonis 1416 CP000140clade_335 N N Parabacteroides goldsteinii 1417 AY974070 clade_335 N NParabacteroides gordonii 1418 AB470344 clade_335 N N Parabacteroides sp.D13 1421 ACPW01000017 clade_335 N N Capnocytophaga genomo sp. 477AY278613 clade_336 N N C1 Capnocytophaga ochracea 480 AEOH01000054clade_336 N N Capnocytophaga sp. GEJ8 481 GU561335 clade_336 N NCapnocytophaga sp. oral strain 486 AY005077 clade_336 N N A47ROYCapnocytophaga sp. S1b 482 U42009 clade_336 N N Paraprevotella clara1426 AFFY01000068 clade_336 N N Bacteroides heparinolyticus 282 JN867284clade_338 N N Prevotella heparinolytica 1500 GQ422742 clade_338 N NTreponema genomo sp. P4 oral 1928 DQ003618 clade_339 N N clone MB2_G19Treponema genomo sp. P6 oral 1930 DQ003625 clade_339 N N clone MB4_G11Treponema sp. oral taxon 254 1952 GU408803 clade_339 N N Treponema sp.oral taxon 508 1956 GU413616 clade_339 N N Treponema sp. oral taxon 5181957 GU413640 clade_339 N N Chlamydia muridarum 502 AE002160 clade_341 NOP Chlamydia trachomatis 504 U68443 clade_341 N OP Chlamydia psittaci503 NR_036864 clade_342 N Category-B Chlamydophila pneumoniae 509NC_002179 clade_342 N OP Chlamydophila psittaci 510 D85712 clade_342 NOP Anaerococcus octavius 146 NR_026360 clade_343 N N Anaerococcus sp.8405254 149 HM587319 clade_343 N N Anaerococcus sp. 9401487 150 HM587322clade_343 N N Anaerococcus sp. 9403502 151 HM587325 clade_343 N NGardnerella vaginalis 923 CP001849 clade_344 N N Campylobacter lari 466CP000932 clade_346 N OP Anaerobiospirillum 142 NR_026075 clade_347 N Nsucciniciproducens Anaerobiospirillum thomasii 143 AJ420985 clade_347 NN Ruminobacter amylophilus 1654 NR_026450 clade_347 N N Succinatimonashippei 1897 AEVO01000027 clade_347 N N Actinomyces europaeus 54NR_026363 clade_348 N N Actinomyces sp. oral clone 82 AY349361 clade_348N N GU009 Moraxella catarrhalis 1260 CP002005 clade_349 N N Moraxellalincolnii 1261 FR822735 clade_349 N N Moraxella sp. 16285 1263 JF682466clade_349 N N Psychrobacter sp. 13983 1613 HM212668 clade_349 N NActinobaculum massiliae 49 AF487679 clade_350 N N Actinobaculum schaalii50 AY957507 clade_350 N N Actinobaculum sp. BM#101342 51 AY282578clade_350 N N Actinobaculum sp. P2P_19 P1 52 AY207066 clade_350 N NActinomyces sp. oral clone 84 AY349363 clade_350 N N IO076 Actinomycessp. oral taxon 93 ACUY01000072 clade_350 N N 848 Actinomyces neuii 65X71862 clade_352 N N Mobiluncus mulieris 1252 ACKW01000035 clade_352 N NBlastomonas natatoria 372 NR_040824 clade_356 N N Novosphingobium 1357AAAV03000008 clade_356 N N aromaticivorans Sphingomonas sp. oral clone1745 AY349411 clade_356 N N FI012 Sphingopyxis alaskensis 1749 CP000356clade_356 N N Oxalobacter formigenes 1389 ACDQ01000020 clade_357 N NVeillonella atypica 1974 AEDS01000059 clade_358 N N Veillonella dispar1975 ACIK02000021 clade_358 N N Veillonella genomo sp. P1 oral 1976DQ003631 clade_358 N N clone MB5_P17 Veillonella parvula 1978ADFU01000009 clade_358 N N Veillonella sp. 3_1_44 1979 ADCV01000019clade_358 N N Veillonella sp. 6_1_27 1980 ADCW01000016 clade_358 N NVeillonella sp. ACP1 1981 HQ616359 clade_358 N N Veillonella sp. AS161982 HQ616365 clade_358 N N Veillonella sp. BS32b 1983 HQ616368clade_358 N N Veillonella sp. ICM51a 1984 HQ616396 clade_358 N NVeillonella sp. MSA12 1985 HQ616381 clade_358 N N Veillonella sp. NVG100cf 1986 EF108443 clade_358 N N Veillonella sp. OK11 1987 JN695650clade_358 N N Veillonella sp. oral clone 1990 AY923144 clade_358 N NASCG01 Veillonella sp. oral clone 1991 AY953257 clade_358 N N ASCG02Veillonella sp. oral clone OH1A 1992 AY947495 clade_358 N N Veillonellasp. oral taxon 158 1993 AENU01000007 clade_358 N N Kocuria marina 1040GQ260086 clade_365 N N Kocuria rhizophila 1042 AY030315 clade_365 N NKocuria rosea 1043 X87756 clade_365 N N Kocuria varians 1044 AF542074clade_365 N N Clostridiaceae bacterium 531 EF451053 clade_368 N N END_2Micrococcus antarcticus 1242 NR_025285 clade_371 N N Micrococcus luteus1243 NR_075062 clade_371 N N Micrococcus lylae 1244 NR_026200 clade_371N N Micrococcus sp. 185 1245 EU714334 clade_371 N N Lactobacillus brevis1072 EU194349 clade_372 N N Lactobacillus parabrevis 1104 NR_042456clade_372 N N Pediococcus acidilactici 1436 ACXB01000026 clade_372 N NPediococcus pentosaceus 1437 NR_075052 clade_372 N N Lactobacillusdextrinicus 1081 NR_036861 clade_373 N N Lactobacillus perolens 1109NR_029360 clade_373 N N Lactobacillus rhamnosus 1113 ABWJ01000068clade_373 N N Lactobacillus saniviri 1118 AB602569 clade_373 N NLactobacillus sp. BT6 1121 HQ616370 clade_373 N N Mycobacteriummageritense 1282 FR798914 clade_374 N OP Mycobacterium neoaurum 1286AF268445 clade_374 N OP Mycobacterium smegmatis 1291 CP000480 clade_374N OP Mycobacterium sp. HE5 1304 AJ012738 clade_374 N N Dysgonomonasgadei 775 ADLV01000001 clade_377 N N Dysgonomonas mossii 776ADLW01000023 clade_377 N N Porphyromonas levii 1474 NR_025907 clade_377N N Porphyromonas somerae 1476 AB547667 clade_377 N N Bacteroidesbarnesiae 267 NR_041446 clade_378 N N Bacteroides coprocola 272ABIY02000050 clade_378 N N Bacteroides coprophilus 273 ACBW01000012clade_378 N N Bacteroides dorei 274 ABWZ01000093 clade_378 N NBacteroides massiliensis 284 AB200226 clade_378 N N Bacteroides plebeius289 AB200218 clade_378 N N Bacteroides sp. 3_1_33FAA 309 ACPS01000085clade_378 N N Bacteroides sp. 3_1_40A 310 ACRT01000136 clade_378 N NBacteroides sp. 4_3_47FAA 313 ACDR02000029 clade_378 N N Bacteroides sp.9_1_42FAA 314 ACAA01000096 clade_378 N N Bacteroides sp. NB_8 323AB117565 clade_378 N N Bacteroides vulgatus 331 CP000139 clade_378 N NBacteroides ovatus 287 ACWH01000036 clade_38 N N Bacteroides sp. 1_1_30294 ADCL01000128 clade_38 N N Bacteroides sp. 2_1_22 297 ACPQ01000117clade_38 N N Bacteroides sp. 2_2_4 299 ABZZ01000168 clade_38 N NBacteroides sp. 3_1_23 308 ACRS01000081 clade_38 N N Bacteroides sp. D1318 ACAB02000030 clade_38 N N Bacteroides sp. D2 321 ACGA01000077clade_38 N N Bacteroides sp. D22 320 ADCK01000151 clade_38 N NBacteroides xylanisolvens 332 ADKP01000087 clade_38 N N Treponemalecithinolyticum 1931 NR_026247 clade_380 N OP Treponema parvum 1933AF302937 clade_380 N OP Treponema sp. oral clone 1940 AY349417 clade_380N N JU025 Treponema sp. oral taxon 270 1954 GQ422733 clade_380 N NParascardovia denticolens 1428 ADEB01000020 clade_381 N N Scardoviainopinata 1688 AB029087 clade_381 N N Scardovia wiggsiae 1689 AY278626clade_381 N N Clostridiales bacterium 533 HM587320 clade_384 N N 9400853Mogibacterium diversum 1254 NR_027191 clade_384 N N Mogibacteriumneglectum 1255 NR_027203 clade_384 N N Mogibacterium pumilum 1256NR_028608 clade_384 N N Mogibacterium timidum 1257 Z36296 clade_384 N NBorrelia burgdorferi 389 ABGI01000001 clade_386 N OP Borrelia garinii392 ABJV01000001 clade_386 N OP Borrelia sp. NE49 397 AJ224142 clade_386N OP Caldimonas manganoxidans 457 NR_040787 clade_387 N N Comamonadaceaebacterium 667 HM099651 clade_387 N N oral taxon F47 Lautropia mirabilis1149 AEQP01000026 clade_387 N N Lautropia sp. oral clone AP009 1150AY005030 clade_387 N N Peptoniphilus asaccharolyticus 1441 D14145clade_389 N N Peptoniphilus duerdenii 1442 EU526290 clade_389 N NPeptoniphilus harei 1443 NR_026358 clade_389 N N Peptoniphilus indolicus1444 AY153431 clade_389 N N Peptoniphilus lacrimalis 1446 ADDO01000050clade_389 N N Peptoniphilus sp. gpac077 1450 AM176527 clade_389 N NPeptoniphilus sp. JC140 1447 JF824803 clade_389 N N Peptoniphilus sp.oral taxon 1452 ADCS01000031 clade_389 N N 386 Peptoniphilus sp. oraltaxon 1453 AEAA01000090 clade_389 N N 836 Peptostreptococcaceae 1454JN837495 clade_389 N N bacterium ph1 Dialister pneumosintes 765 HM596297clade_390 N N Dialister sp. oral taxon 502 767 GQ422739 clade_390 N NCupriavidus metallidurans 741 GU230889 clade_391 N N Herbaspirillumseropedicae 1001 CP002039 clade_391 N N Herbaspirillum sp. JC206 1002JN657219 clade_391 N N Janthinobacterium sp. SY12 1015 EF455530clade_391 N N Massilia sp. CCUG 43427A 1197 FR773700 clade_391 N NRalstonia pickettii 1615 NC_010682 clade_391 N N Ralstonia sp. 5_7_47FAA1616 ACUF01000076 clade_391 N N Francisella novicida 889 ABSS01000002clade_392 N N Francisella philomiragia 890 AY928394 clade_392 N NFrancisella tularensis 891 ABAZ01000082 clade_392 N Category-AIgnatzschineria indica 1009 HQ823562 clade_392 N N Ignatzschineria sp.NML 1010 HQ823559 clade_392 N N 95_0260 Streptococcus mutans 1814AP010655 clade_394 N N Lactobacillus gasseri 1084 ACOZ01000018 clade_398N N Lactobacillus hominis 1090 FR681902 clade_398 N N Lactobacillusiners 1091 AEKJ01000002 clade_398 N N Lactobacillus johnsonii 1093AE017198 clade_398 N N Lactobacillus senioris 1119 AB602570 clade_398 NN Lactobacillus sp. oral clone 1135 AY349382 clade_398 N N HT002Weissella beninensis 2006 EU439435 clade_398 N N Sphingomonas echinoides1744 NR_024700 clade_399 N N Sphingomonas sp. oral taxon 1747 HM099639clade_399 N N A09 Sphingomonas sp. oral taxon 1748 HM099645 clade_399 NN F71 Zymomonas mobilis 2032 NR_074274 clade_399 N N Arcanobacterium 174NR_025347 clade_400 N N haemolyticum Arcanobacterium pyogenes 175GU585578 clade_400 N N Trueperella pyogenes 1962 NR_044858 clade_400 N NLactococcus garvieae 1144 AF061005 clade_401 N N Lactococcus lactis 1145CP002365 clade_401 N N Brevibacterium mcbrellneri 424 ADNU01000076clade_402 N N Brevibacterium paucivorans 425 EU086796 clade_402 N NBrevibacterium sp. JC43 428 JF824806 clade_402 N N Selenomonas artemidis1692 HM596274 clade_403 N N Selenomonas sp. FOBRC9 1704 HQ616378clade_403 N N Selenomonas sp. oral taxon 1715 AENV01000007 clade_403 N N137 Desmospora activa 751 AM940019 clade_404 N N Desmospora sp. 8437 752AFHT01000143 clade_404 N N Paenibacillus sp. oral taxon 1407 HM099647clade_404 N N F45 Corynebacterium 682 ADNS01000011 clade_405 N Nammoniagenes Corynebacterium 687 ACLH01000041 clade_405 N N aurimucosumCorynebacterium bovis 688 AF537590 clade_405 N N Corynebacterium canis689 GQ871934 clade_405 N N Corynebacterium casei 690 NR_025101 clade_405N N Corynebacterium durum 694 Z97069 clade_405 N N Corynebacteriumefficiens 695 ACLI01000121 clade_405 N N Corynebacterium falsenii 696Y13024 clade_405 N N Corynebacterium flavescens 697 NR_037040 clade_405N N Corynebacterium glutamicum 701 BA000036 clade_405 N NCorynebacterium jeikeium 704 ACYW01000001 clade_405 N OP Corynebacterium705 NR_026380 clade_405 N N kroppenstedtii Corynebacterium 706ACHJ01000075 clade_405 N N lipophiloflavum Corynebacterium matruchotii709 ACSH02000003 clade_405 N N Corynebacterium 710 X82064 clade_405 N Nminutissimum Corynebacterium resistens 718 ADGN01000058 clade_405 N NCorynebacterium simulans 720 AF537604 clade_405 N N Corynebacteriumsingulare 721 NR_026394 clade_405 N N Corynebacterium sp. 1 ex 722Y13427 clade_405 N N sheep Corynebacterium sp. NML 726 GU238413clade_405 N N 99_0018 Corynebacterium striatum 727 ACGE01000001clade_405 N OP Corynebacterium urealyticum 732 X81913 clade_405 N OPCorynebacterium variabile 734 NR_025314 clade_405 N N Aerococcussanguinicola 98 AY837833 clade_407 N N Aerococcus urinae 99 CP002512clade_407 N N Aerococcus urinaeequi 100 NR_043443 clade_407 N NAerococcus viridans 101 ADNT01000041 clade_407 N N Fusobacteriumnaviforme 898 HQ223106 clade_408 N N Moryella indoligenes 1268 AF527773clade_408 N N Selenomonas genomo sp. P5 1697 AY341820 clade_410 N NSelenomonas sp. oral clone 1710 AY349408 clade_410 N N IQ048 Selenomonassputigena 1717 ACKP02000033 clade_410 N N Hyphomicrobium sulfonivorans1007 AY468372 clade_411 N N Methylocella silvestris 1228 NR_074237clade_411 N N Legionella pneumophila 1153 NC_002942 clade_412 N OPLactobacillus coryniformis 1077 NR_044705 clade_413 N N Arthrobacteragilis 178 NR_026198 clade_414 N N Arthrobacter arilaitensis 179NR_074608 clade_414 N N Arthrobacter bergerei 180 NR_025612 clade_414 NN Arthrobacter globiformis 181 NR_026187 clade_414 N N Arthrobacternicotianae 182 NR_026190 clade_414 N N Mycobacterium abscessus 1269AGQU01000002 clade_418 N OP Mycobacterium chelonae 1273 AB548610clade_418 N OP Bacteroides salanitronis 291 CP002530 clade_419 N NParaprevotella xylaniphila 1427 AFBR01000011 clade_419 N N Barnesiellaintestinihominis 336 AB370251 clade_420 N N Barnesiella viscericola 337NR_041508 clade_420 N N Parabacteroides sp. NS31_3 1422 JN029805clade_420 N N Porphyromonadaceae 1470 EF184292 clade_420 N N bacteriumNML 060648 Tannerella forsythia 1913 CP003191 clade_420 N N Tannerellasp. 1914 ACWX01000068 clade_420 N N 6_1_58FAA_CT1 Mycoplasmaamphoriforme 1311 AY531656 clade_421 N N Mycoplasma genitalium 1317L43967 clade_421 N N Mycoplasma pneumoniae 1322 NC_000912 clade_421 N NMycoplasma penetrans 1321 NC_004432 clade_422 N N Ureaplasma parvum 1966AE002127 clade_422 N N Ureaplasma urealyticum 1967 AAYN01000002clade_422 N N Treponema genomo sp. P1 1927 AY341822 clade_425 N NTreponema sp. oral taxon 228 1943 GU408580 clade_425 N N Treponema sp.oral taxon 230 1944 GU408603 clade_425 N N Treponema sp. oral taxon 2311945 GU408631 clade_425 N N Treponema sp. oral taxon 232 1946 GU408646clade_425 N N Treponema sp. oral taxon 235 1947 GU408673 clade_425 N NTreponema sp. ovine footrot 1959 AJ010951 clade_425 N N Treponemavincentii 1960 ACYH01000036 clade_425 N OP Burkholderiales bacterium 452ADCQ01000066 clade_432 N OP 1_1_47 Parasutterella 1429 AFBP01000029clade_432 N N excrementihominis Parasutterella secunda 1430 AB491209clade_432 N N Sutterella morbirenis 1898 AJ832129 clade_432 N NSutterella sanguinus 1900 AJ748647 clade_432 N N Sutterella sp. YIT12072 1901 AB491210 clade_432 N N Sutterella stercoricanis 1902NR_025600 clade_432 N N Sutterella wadsworthensis 1903 ADMF01000048clade_432 N N Propionibacterium 1572 NR_036972 clade_433 N Nfreudenreichii Propionibacterium sp. oral 1580 GQ422728 clade_433 N Ntaxon 192 Tessaracoccus sp. oral taxon 1917 HM099640 clade_433 N N F04Peptoniphilus ivorii 1445 Y07840 clade_434 N N Peptoniphilus sp. gpac0071448 AM176517 clade_434 N N Peptoniphilus sp. gpac018A 1449 AM176519clade_434 N N Peptoniphilus sp. gpac148 1451 AM176535 clade_434 N NFlexispira rappini 887 AY126479 clade_436 N N Helicobacter bilis 993ACDN01000023 clade_436 N N Helicobacter cinaedi 995 ABQT01000054clade_436 N N Helicobacter sp. None 998 U44756 clade_436 N NBrevundimonas subvibrioides 429 CP002102 clade_438 N N Hyphomonasneptunium 1008 NR_074092 clade_438 N N Phenylobacterium zucineum 1465AY628697 clade_438 N N Streptococcus downei 1793 AEKN01000002 clade_441N N Streptococcus sp. SHV515 1848 Y07601 clade_441 N N Acinetobacter sp.CIP 53.82 40 JQ638584 clade_443 N N Halomonas elongata 990 NR_074782clade_443 N N Halomonas johnsoniae 991 FR775979 clade_443 N NButyrivibrio fibrisolvens 456 U41172 clade_444 N N Roseburia sp. 11SE371640 FM954975 clade_444 N N Roseburia sp. 11SE38 1641 FM954976 clade_444N N Shuttleworthia satelles 1728 ACIP02000004 clade_444 N NShuttleworthia sp. MSX8B 1729 HQ616383 clade_444 N N Shuttleworthia sp.oral taxon 1730 GU432167 clade_444 N N G69 Bdellovibrio sp. MPA 344AY294215 clade_445 N N Desulfobulbus sp. oral clone 755 AY005036clade_445 N N CH031 Desulfovibrio desulfuricans 757 DQ092636 clade_445 NN Desulfovibrio fairfieldensis 758 U42221 clade_445 N N Desulfovibriopiger 759 AF192152 clade_445 N N Desulfovibrio sp. 3_1_syn3 760ADDR01000239 clade_445 N N Geobacter bemidjiensis 941 CP001124 clade_445N N Brachybacterium alimentarium 401 NR_026269 clade_446 N NBrachybacterium 402 AB537169 clade_446 N N conglomeratum Brachybacterium403 NR_026272 clade_446 N N tyrofermentans Dermabacter hominis 749FJ263375 clade_446 N N Aneurinibacillus 171 NR_029303 clade_448 N Nthermoaerophilus Brevibacillus agri 409 NR_040983 clade_448 N NBrevibacillus centrosporus 411 NR_043414 clade_448 N N Brevibacilluschoshinensis 412 NR_040980 clade_448 N N Brevibacillus invocatus 413NR_041836 clade_448 N N Brevibacillus parabrevis 415 NR_040981 clade_448N N Brevibacillus reuszeri 416 NR_040982 clade_448 N N Brevibacillus sp.phR 417 JN837488 clade_448 N N Brevibacillus thermoruber 418 NR_026514clade_448 N N Lactobacillus murinus 1100 NR_042231 clade_449 N NLactobacillus oeni 1102 NR_043095 clade_449 N N Lactobacillus ruminis1115 ACGS02000043 clade_449 N N Lactobacillus vini 1141 NR_042196clade_449 N N Gemella haemolysans 924 ACDZ02000012 clade_450 N N Gemellamorbillorum 925 NR_025904 clade_450 N N Gemella morbillorum 926ACRX01000010 clade_450 N N Gemella sanguinis 927 ACRY01000057 clade_450N N Gemella sp. oral clone 929 AY923133 clade_450 N N ASCE02 Gemella sp.oral clone 930 AY923139 clade_450 N N ASCF04 Gemella sp. oral clone 931AY923143 clade_450 N N ASCF12 Gemella sp. WAL 1945J 928 EU427463clade_450 N N Sporolactobacillus nakayamae 1753 NR_042247 clade_451 N NGluconacetobacter entanii 945 NR_028909 clade_452 N N Gluconacetobactereuropaeus 946 NR_026513 clade_452 N N Gluconacetobacter hansenii 947NR_026133 clade_452 N N Gluconacetobacter oboediens 949 NR_041295clade_452 N N Gluconacetobacter xylinus 950 NR_074338 clade_452 N NAuritibacter ignavus 193 FN554542 clade_453 N N Dermacoccus sp. Ellin185750 AEIQ01000090 clade_453 N N Janibacter limosus 1013 NR_026362clade_453 N N Janibacter melonis 1014 EF063716 clade_453 N N Acetobacteraceti 7 NR_026121 clade_454 N N Acetobacter fabarum 8 NR_042678clade_454 N N Acetobacter lovaniensis 9 NR_040832 clade_454 N NAcetobacter malorum 10 NR_025513 clade_454 N N Acetobacter orientalis 11NR_028625 clade_454 N N Acetobacter pasteurianus 12 NR_026107 clade_454N N Acetobacter pomorum 13 NR_042112 clade_454 N N Acetobacter syzygii14 NR_040868 clade_454 N N Acetobacter tropicalis 15 NR_036881 clade_454N N Gluconacetobacter 943 NR_028767 clade_454 N N azotocaptansGluconacetobacter 944 NR_074292 clade_454 N N diazotrophicusGluconacetobacter johannae 948 NR_024959 clade_454 N N Nocardiabrasiliensis 1351 AIHV01000038 clade_455 N N Nocardia cyriacigeorgica1352 HQ009486 clade_455 N N Nocardia puris 1354 NR_028994 clade_455 N NNocardia sp. 01_Je_025 1355 GU574059 clade_455 N N Rhodococcus equi 1623ADNW01000058 clade_455 N N Oceanobacillus caeni 1358 NR_041533 clade_456N N Oceanobacillus sp. Ndiop 1359 CAER01000083 clade_456 N NOrnithinibacillus bavariensis 1384 NR_044923 clade_456 N NOrnithinibacillus sp. 7_10AIA 1385 FN397526 clade_456 N N Virgibacillusproomii 2005 NR_025308 clade_456 N N Corynebacterium amycolatum 683ABZU01000033 clade_457 N OP Corynebacterium hansenii 702 AM946639clade_457 N N Corynebacterium xerosis 735 FN179330 clade_457 N OPStaphylococcaceae bacterium 1756 AY841362 clade_458 N N NML 92_0017Staphylococcus fleurettii 1766 NR_041326 clade_458 N N Staphylococcussciuri 1774 NR_025520 clade_458 N N Staphylococcus vitulinus 1779NR_024670 clade_458 N N Stenotrophomonas maltophilia 1782 AAVZ01000005clade_459 N N Stenotrophomonas sp. FG_6 1783 EF017810 clade_459 N NMycobacterium africanum 1270 AF480605 clade_46 N OP Mycobacteriumalsiensis 1271 AJ938169 clade_46 N OP Mycobacterium avium 1272 CP000479clade_46 N OP Mycobacterium colombiense 1274 AM062764 clade_46 N OPMycobacterium gordonae 1276 GU142930 clade_46 N OP Mycobacteriumintracellulare 1277 GQ153276 clade_46 N OP Mycobacterium kansasii 1278AF480601 clade_46 N OP Mycobacterium lacus 1279 NR_025175 clade_46 N OPMycobacterium leprae 1280 FM211192 clade_46 N OP Mycobacteriumlepromatosis 1281 EU203590 clade_46 N OP Mycobacterium mantenii 1283FJ042897 clade_46 N OP Mycobacterium marinum 1284 NC_010612 clade_46 NOP Mycobacterium microti 1285 NR_025234 clade_46 N OP Mycobacterium 1287ADNV01000350 clade_46 N OP parascrofulaceum Mycobacterium seoulense 1290DQ536403 clade_46 N OP Mycobacterium sp. 1761 1292 EU703150 clade_46 N NMycobacterium sp. 1791 1295 EU703148 clade_46 N N Mycobacterium sp. 17971296 EU703149 clade_46 N N Mycobacterium sp. 1298 HQ174245 clade_46 N NB10_07.09.0206 Mycobacterium sp. 1305 HM627011 clade_46 N N NLA001000736Mycobacterium sp. W 1306 DQ437715 clade_46 N N Mycobacteriumtuberculosis 1307 CP001658 clade_46 N Category-C Mycobacterium ulcerans1308 AB548725 clade_46 N OP Mycobacterium vulneris 1309 EU834055clade_46 N OP Xanthomonas campestris 2016 EF101975 clade_461 N NXanthomonas sp. kmd_489 2017 EU723184 clade_461 N N Dietzianatronolimnaea 769 GQ870426 clade_462 N N Dietzia sp. BBDP51 770DQ337512 clade_462 N N Dietzia sp. CA149 771 GQ870422 clade_462 N NDietzia timorensis 772 GQ870424 clade_462 N N Gordonia bronchialis 951NR_027594 clade_463 N N Gordonia polyisoprenivorans 952 DQ385609clade_463 N N Gordonia sp. KTR9 953 DQ068383 clade_463 N N Gordoniasputi 954 FJ536304 clade_463 N N Gordonia terrae 955 GQ848239 clade_463N N Leptotrichia goodfellowii 1167 ADAD01000110 clade_465 N NLeptotrichia sp. oral clone 1174 AY349387 clade_465 N N IK040Leptotrichia sp. oral clone 1175 AY207053 clade_465 N N P2PB_51 P1Bacteroidales genomo sp. P7 264 DQ003623 clade_466 N N oral cloneMB3_P19 Butyricimonas virosa 454 AB443949 clade_466 N N Odoribacterlaneus 1363 AB490805 clade_466 N N Odoribacter splanchnicus 1364CP002544 clade_466 N N Capnocytophaga gingivalis 478 ACLQ01000011clade_467 N N Capnocytophaga granulosa 479 X97248 clade_467 N NCapnocytophaga sp. oral clone 483 AY005074 clade_467 N N AH015Capnocytophaga sp. oral strain 487 AY005073 clade_467 N N S3Capnocytophaga sp. oral taxon 488 AEXX01000050 clade_467 N N 338Capnocytophaga canimorsus 476 CP002113 clade_468 N N Capnocytophaga sp.oral clone 485 AY349368 clade_468 N N ID062 Lactobacillus catenaformis1075 M23729 clade_469 N N Lactobacillus vitulinus 1142 NR_041305clade_469 N N Cetobacterium somerae 501 AJ438155 clade_470 N NFusobacterium gonidiaformans 896 ACET01000043 clade_470 N NFusobacterium mortiferum 897 ACDB02000034 clade_470 N N Fusobacteriumnecrogenes 899 X55408 clade_470 N N Fusobacterium necrophorum 900AM905356 clade_470 N N Fusobacterium sp. 12_1B 905 AGWJ01000070clade_470 N N Fusobacterium sp. 3_1_5R 911 ACDD01000078 clade_470 N NFusobacterium sp. D12 918 ACDG02000036 clade_470 N N Fusobacteriumulcerans 921 ACDH01000090 clade_470 N N Fusobacterium varium 922ACIE01000009 clade_470 N N Mycoplasma arthritidis 1312 NC_011025clade_473 N N Mycoplasma faucium 1314 NR_024983 clade_473 N N Mycoplasmahominis 1318 AF443616 clade_473 N N Mycoplasma orale 1319 AY796060clade_473 N N Mycoplasma salivarium 1324 M24661 clade_473 N NMitsuokella jalaludinii 1247 NR_028840 clade_474 N N Mitsuokellamultacida 1248 ABWK02000005 clade_474 N N Mitsuokella sp. oral taxon 5211249 GU413658 clade_474 N N Mitsuokella sp. oral taxon G68 1250 GU432166clade_474 N N Selenomonas genomo sp. C1 1695 AY278627 clade_474 N NSelenomonas genomo sp. P8 1700 DQ003628 clade_474 N N oral clone MB5_P06Selenomonas ruminantium 1703 NR_075026 clade_474 N N Veillonellaceaebacterium oral 1994 GU402916 clade_474 N N taxon 131 Alloscardoviaomnicolens 139 NR_042583 clade_475 N N Alloscardovia sp. OB7196 140AB425070 clade_475 N N Bifidobacterium urinalis 366 AJ278695 clade_475 NN Prevotella loescheii 1503 JN867231 clade_48 N N Prevotella sp. oralclone 1530 DQ272511 clade_48 N N ASCG12 Prevotella sp. oral clone 1540AY349398 clade_48 N N GU027 Prevotella sp. oral taxon 472 1553ACZS01000106 clade_48 N N Selenomonas dianae 1693 GQ422719 clade_480 N NSelenomonas flueggei 1694 AF287803 clade_480 N N Selenomonas genomo sp.C2 1696 AY278628 clade_480 N N Selenomonas genomo sp. P6 1698 DQ003636clade_480 N N oral clone MB3_C41 Selenomonas genomo sp. P7 1699 DQ003627clade_480 N N oral clone MB5_C08 Selenomonas infelix 1701 AF287802clade_480 N N Selenomonas noxia 1702 GU470909 clade_480 N N Selenomonassp. oral clone 1705 AY349403 clade_480 N N FT050 Selenomonas sp. oralclone 1706 AY349404 clade_480 N N GI064 Selenomonas sp. oral clone 1707AY349405 clade_480 N N GT010 Selenomonas sp. oral clone 1708 AY349406clade_480 N N HU051 Selenomonas sp. oral clone 1709 AY349407 clade_480 NN IK004 Selenomonas sp. oral clone 1711 AY349409 clade_480 N N JI021Selenomonas sp. oral clone 1712 AY349410 clade_480 N N JS031 Selenomonassp. oral clone 1713 AY947498 clade_480 N N OH4A Selenomonas sp. oralclone 1714 AY207052 clade_480 N N P2PA_80 P4 Selenomonas sp. oral taxon1716 AEEJ01000007 clade_480 N N 149 Veillonellaceae bacterium oral 1995GU470897 clade_480 N N taxon 155 Agrococcus jenensis 117 NR_026275clade_484 N N Microbacterium gubbeenense 1232 NR_025098 clade_484 N NPseudoclavibacter sp. Timone 1590 FJ375951 clade_484 N N Tropherymawhipplei 1961 BX251412 clade_484 N N Zimmermannella bifida 2031 AB012592clade_484 N N Legionella hackeliae 1151 M36028 clade_486 N OP Legionellalongbeachae 1152 M36029 clade_486 N OP Legionella sp. D3923 1154JN380999 clade_486 N OP Legionella sp. D4088 1155 JN381012 clade_486 NOP Legionella sp. H63 1156 JF831047 clade_486 N OP Legionella sp. NML93L054 1157 GU062706 clade_486 N OP Legionella steelei 1158 HQ398202clade_486 N OP Tatlockia micdadei 1915 M36032 clade_486 N N Helicobacterpullorum 996 ABQU01000097 clade_489 N N Acetobacteraceae bacterium 16AGEZ01000040 clade_490 N N AT_5844 Roseomonas cervicalis 1643ADVL01000363 clade_490 N N Roseomonas mucosa 1644 NR_028857 clade_490 NN Roseomonas sp. NML94_0193 1645 AF533357 clade_490 N N Roseomonas sp.NML97_0121 1646 AF533359 clade_490 N N Roseomonas sp. NML98_0009 1647AF533358 clade_490 N N Roseomonas sp. NML98_0157 1648 AF533360 clade_490N N Rickettsia akari 1627 CP000847 clade_492 N OP Rickettsia conorii1628 AE008647 clade_492 N OP Rickettsia prowazekii 1629 M21789 clade_492N Category-B Rickettsia rickettsii 1630 NC_010263 clade_492 N OPRickettsia slovaca 1631 L36224 clade_492 N OP Rickettsia typhi 1632AE017197 clade_492 N OP Anaeroglobus geminatus 160 AGCJ01000054clade_493 N N Megasphaera genomo sp. C1 1201 AY278622 clade_493 N NMegasphaera micronuciformis 1203 AECS01000020 clade_493 N NClostridiales genomo sp. 540 CP001850 clade_495 N N BVAB3 Tsukamurellapaurometabola 1963 X80628 clade_496 N N Tsukamurella tyrosinosolvens1964 AB478958 clade_496 N N Abiotrophia para_adiacens 2 AB022027clade_497 N N Carnobacterium divergens 492 NR_044706 clade_497 N NCarnobacterium 493 NC_019425 clade_497 N N maltaromaticum Enterococcusavium 800 AF133535 clade_497 N N Enterococcus caccae 801 AY943820clade_497 N N Enterococcus casseliflavus 802 AEWT01000047 clade_497 N NEnterococcus durans 803 AJ276354 clade_497 N N Enterococcus faecalis 804AE016830 clade_497 N N Enterococcus faecium 805 AM157434 clade_497 N NEnterococcus gallinarum 806 AB269767 clade_497 N N Enterococcus gilvus807 AY033814 clade_497 N N Enterococcus hawaiiensis 808 AY321377clade_497 N N Enterococcus hirae 809 AF061011 clade_497 N N Enterococcusitalicus 810 AEPV01000109 clade_497 N N Enterococcus mundtii 811NR_024906 clade_497 N N Enterococcus raffinosus 812 FN600541 clade_497 NN Enterococcus sp. BV2CASA2 813 JN809766 clade_497 N N Enterococcus sp.CCRI_16620 814 GU457263 clade_497 N N Enterococcus sp. F95 815 FJ463817clade_497 N N Enterococcus sp. RfL6 816 AJ133478 clade_497 N NEnterococcus thailandicus 817 AY321376 clade_497 N N Fusobacteriumcanifelinum 893 AY162222 clade_497 N N Fusobacterium genomo sp. C1 894AY278616 clade_497 N N Fusobacterium genomo sp. C2 895 AY278617clade_497 N N Fusobacterium periodonticum 902 ACJY01000002 clade_497 N NFusobacterium sp. 1_1_41FAA 906 ADGG01000053 clade_497 N N Fusobacteriumsp. 11_3_2 904 ACUO01000052 clade_497 N N Fusobacterium sp. 2_1_31 907ACDC02000018 clade_497 N N Fusobacterium sp. 3_1_27 908 ADGF01000045clade_497 N N Fusobacterium sp. 3_1_33 909 ACQE01000178 clade_497 N NFusobacterium sp. 3_1_36A2 910 ACPU01000044 clade_497 N N Fusobacteriumsp. AC18 912 HQ616357 clade_497 N N Fusobacterium sp. ACB2 913 HQ616358clade_497 N N Fusobacterium sp. AS2 914 HQ616361 clade_497 N NFusobacterium sp. CM1 915 HQ616371 clade_497 N N Fusobacterium sp. CM21916 HQ616375 clade_497 N N Fusobacterium sp. CM22 917 HQ616376 clade_497N N Fusobacterium sp. oral clone 919 AY923141 clade_497 N N ASCF06Fusobacterium sp. oral clone 920 AY953256 clade_497 N N ASCF11Granulicatella adiacens 959 ACKZ01000002 clade_497 N N Granulicatellaelegans 960 AB252689 clade_497 N N Granulicatella paradiacens 961AY879298 clade_497 N N Granulicatella sp. oral clone 963 AY923126clade_497 N N ASC02 Granulicatella sp. oral clone 964 DQ341469 clade_497N N ASCA05 Granulicatella sp. oral clone 965 AY953251 clade_497 N NASCB09 Granulicatella sp. oral clone 966 AY923146 clade_497 N N ASCG05Tetragenococcus halophilus 1918 NR_075020 clade_497 N N Tetragenococcuskoreensis 1919 NR_043113 clade_497 N N Vagococcus fluvialis 1973NR_026489 clade_497 N N Chryseobacterium anthropi 514 AM982793 clade_498N N Chryseobacterium gleum 515 ACKQ02000003 clade_498 N NChryseobacterium hominis 516 NR_042517 clade_498 N N Treponemarefringens 1936 AF426101 clade_499 N OP Treponema sp. oral clone 1941AY349416 clade_499 N N JU031 Treponema sp. oral taxon 239 1948 GU408738clade_499 N N Treponema sp. oral taxon 271 1955 GU408871 clade_499 N NAlistipes finegoldii 129 NR_043064 clade_500 N N Alistipes onderdonkii131 NR_043318 clade_500 N N Alistipes putredinis 132 ABFK02000017clade_500 N N Alistipes shahii 133 FP929032 clade_500 N N Alistipes sp.HGB5 134 AENZ01000082 clade_500 N N Alistipes sp. JC50 135 JF824804clade_500 N N Alistipes sp. RMA 9912 136 GQ140629 clade_500 N NMycoplasma agalactiae 1310 AF010477 clade_501 N N Mycoplasma bovoculi1313 NR_025987 clade_501 N N Mycoplasma fermentans 1315 CP002458clade_501 N N Mycoplasma flocculare 1316 X62699 clade_501 N N Mycoplasmaovipneumoniae 1320 NR_025989 clade_501 N N Arcobacter butzleri 176AEPT01000071 clade_502 N N Arcobacter cryaerophilus 177 NR_025905clade_502 N N Campylobacter curvus 461 NC_009715 clade_502 N OPCampylobacter rectus 467 ACFU01000050 clade_502 N OP Campylobactershowae 468 ACVQ01000030 clade_502 N OP Campylobacter sp. FOBRC14 469HQ616379 clade_502 N OP Campylobacter sp. FOBRC15 470 HQ616380 clade_502N OP Campylobacter sp. oral clone 471 AY005038 clade_502 N OP BB120Campylobacter sputorum 472 NR_044839 clade_502 N OP Bacteroidesureolyticus 330 GQ167666 clade_504 N N Campylobacter gracilis 463ACYG01000026 clade_504 N OP Campylobacter hominis 464 NC_009714clade_504 N OP Dialister invisus 762 ACIM02000001 clade_506 N NDialister micraerophilus 763 AFBB01000028 clade_506 N N Dialistermicroaerophilus 764 AENT01000008 clade_506 N N Dialisterpropionicifaciens 766 NR_043231 clade_506 N N Dialister succinatiphilus768 AB370249 clade_506 N N Megasphaera elsdenii 1200 AY038996 clade_506N N Megasphaera genomo sp. 1202 ADGP01000010 clade_506 N N type_1Megasphaera sp. BLPYG_07 1204 HM990964 clade_506 N N Megasphaera sp.UPII 199_6 1205 AFIJ01000040 clade_506 N N Chromobacterium violaceum 513NC_005085 clade_507 N N Laribacter hongkongensis 1148 CP001154 clade_507N N Methylophilus sp. ECd5 1229 AY436794 clade_507 N N Finegoldia magna883 ACHM02000001 clade_509 N N Parvimonas micra 1431 AB729072 clade_509N N Parvimonas sp. oral taxon 110 1432 AFII01000002 clade_509 N NPeptostreptococcus micros 1456 AM176538 clade_509 N N Peptostreptococcussp. oral 1460 AY349390 clade_509 N N clone FJ023 Peptostreptococcus sp.1458 AY207059 clade_509 N N P4P_31 P3 Helicobacter pylori 997 CP000012clade_510 N OP Anaplasma marginale 165 ABOR01000019 clade_511 N NAnaplasma phagocytophilum 166 NC_007797 clade_511 N N Ehrlichiachaffeensis 783 AAIF01000035 clade_511 N OP Neorickettsia risticii 1349CP001431 clade_511 N N Neorickettsia sennetsu 1350 NC_007798 clade_511 NN Pseudoramibacter alactolyticus 1606 AB036759 clade_512 N N Veillonellamontpellierensis 1977 AF473836 clade_513 N N Veillonella sp. oral clone1988 AY923118 clade_513 N N ASCA08 Veillonella sp. oral clone 1989AY923122 clade_513 N N ASCB03 Inquilinus limosus 1012 NR_029046clade_514 N N Sphingomonas sp. oral clone 1746 AY349412 clade_514 N NFZ016 Anaerococcus lactolyticus 145 ABYO01000217 clade_515 N NAnaerococcus prevotii 147 CP001708 clade_515 N N Anaerococcus sp.gpac104 152 AM176528 clade_515 N N Anaerococcus sp. gpac126 153 AM176530clade_515 N N Anaerococcus sp. gpac155 154 AM176536 clade_515 N NAnaerococcus sp. gpac199 155 AM176539 clade_515 N N Anaerococcustetradius 157 ACGC01000107 clade_515 N N Bacteroides coagulans 271AB547639 clade_515 N N Clostridiales bacterium 534 HM587324 clade_515 NN 9403326 Clostridiales bacterium ph2 539 JN837487 clade_515 N NPeptostreptococcus sp. 9succ1 1457 X90471 clade_515 N NPeptostreptococcus sp. oral 1459 AB175072 clade_515 N N clone AP24Tissierella praeacuta 1924 NR_044860 clade_515 N N Helicobactercanadensis 994 ABQS01000108 clade_518 N N Peptostreptococcus 1455AY326462 clade_520 N N anaerobius Peptostreptococcus stomatis 1461ADGQ01000048 clade_520 N N Bilophila wadsworthia 367 ADCP01000166clade_521 N N Desulfovibrio vulgaris 761 NR_074897 clade_521 N NActinomyces nasicola 64 AJ508455 clade_523 N N Cellulosimicrobium funkei500 AY501364 clade_523 N N Lactococcus raffinolactis 1146 NR_044359clade_524 N N Bacteroidales genomo sp. P1 258 AY341819 clade_529 N NBacteroidales genomo sp. P2 259 DQ003613 clade_529 N N oral cloneMB1_G13 Bacteroidales genomo sp. P3 260 DQ003615 clade_529 N N oralclone MB1_G34 Bacteroidales genomo sp. P4 261 DQ003617 clade_529 N Noral clone MB2_G17 Bacteroidales genomo sp. P5 262 DQ003619 clade_529 NN oral clone MB2_P04 Bacteroidales genomo sp. P6 263 DQ003634 clade_529N N oral clone MB3_C19 Bacteroidales genomo sp. P8 265 DQ003626clade_529 N N oral clone MB4_G15 Bacteroidetes bacterium oral 333HM099638 clade_530 N N taxon D27 Bacteroidetes bacterium oral 334HM099643 clade_530 N N taxon F31 Bacteroidetes bacterium oral 335HM099649 clade_530 N N taxon F44 Flavobacterium sp. NF2_1 885 FJ195988clade_530 N N Myroides odoratimimus 1326 NR_042354 clade_530 N NMyroides sp. MY15 1327 GU253339 clade_530 N N Chlamydiales bacteriumNS16 507 JN606076 clade_531 N N Chlamydophila pecorum 508 D88317clade_531 N OP Parachlamydia sp. UWE25 1423 BX908798 clade_531 N NFusobacterium russii 903 NR_044687 clade_532 N N Streptobacillusmoniliformis 1784 NR_027615 clade_532 N N Eubacteriaceae bacterium 833AY207060 clade_533 N N P4P_50 P4 Abiotrophia defectiva 1 ACIN02000016clade_534 N N Abiotrophia sp. oral clone 3 AY207063 clade_534 N NP4PA_155 P1 Catonella genomo sp. P1 oral 496 DQ003629 clade_534 N Nclone MB5_P12 Catonella morbi 497 ACIL02000016 clade_534 N N Catonellasp. oral clone FL037 498 AY349369 clade_534 N N Eremococcus coleocola818 AENN01000008 clade_534 N N Facklamia hominis 879 Y10772 clade_534 NN Granulicatella sp. M658_99_3 962 AJ271861 clade_534 N N Campylobactercoli 459 AAFL01000004 clade_535 N OP Campylobacter concisus 460 CP000792clade_535 N OP Campylobacter fetus 462 ACLG01001177 clade_535 N OPCampylobacter jejuni 465 AL139074 clade_535 N Category-B Campylobacterupsaliensis 473 AEPU01000040 clade_535 N OP Atopobium minutum 183HM007583 clade_539 N N Atopobium parvulum 184 CP001721 clade_539 N NAtopobium rimae 185 ACFE01000007 clade_539 N N Atopobium sp. BS2 186HQ616367 clade_539 N N Atopobium sp. F0209 187 EU592966 clade_539 N NAtopobium sp. ICM42b10 188 HQ616393 clade_539 N N Atopobium sp. ICM57189 HQ616400 clade_539 N N Atopobium vaginae 190 AEDQ01000024 clade_539N N Coriobacteriaceae bacterium 677 JN809768 clade_539 N N BV3Ac1Actinomyces naeslundii 63 X81062 clade_54 N N Actinomyces oricola 67NR_025559 clade_54 N N Actinomyces oris 69 BABV01000070 clade_54 N NActinomyces sp. 7400942 70 EU484334 clade_54 N N Actinomyces sp. ChDCB197 72 AF543275 clade_54 N N Actinomyces sp. GEJ15 73 GU561313 clade_54N N Actinomyces sp. M2231_94_1 79 AJ234063 clade_54 N N Actinomyces sp.oral clone 83 AY349362 clade_54 N N GU067 Actinomyces sp. oral clone 85AY349364 clade_54 N N IO077 Actinomyces sp. oral clone 86 AY349365clade_54 N N IP073 Actinomyces sp. oral clone 88 AY349367 clade_54 N NJA063 Actinomyces sp. oral taxon 89 AFBL01000010 clade_54 N N 170Actinomyces sp. oral taxon 90 AECW01000034 clade_54 N N 171 Actinomycesurogenitalis 95 ACFH01000038 clade_54 N N Actinomyces viscosus 96ACRE01000096 clade_54 N N Orientia tsutsugamushi 1383 AP008981 clade_541N OP Megamonas funiformis 1198 AB300988 clade_542 N N Megamonashypermegale 1199 AJ420107 clade_542 N N Aeromicrobium marinum 102NR_025681 clade_544 N N Aeromicrobium sp. JC14 103 JF824798 clade_544 NN Luteococcus sanguinis 1190 NR_025507 clade_544 N NPropionibacteriaceae 1568 EF599122 clade_544 N N bacterium NML 02_0265Rhodococcus 1622 X80615 clade_546 N N corynebacterioides Rhodococcuserythropolis 1624 ACNO01000030 clade_546 N N Rhodococcus fascians 1625NR_037021 clade_546 N N Segniliparus rotundus 1690 CP001958 clade_546 NN Segniliparus rugosus 1691 ACZI01000025 clade_546 N N Exiguobacteriumacetylicum 878 FJ970034 clade_547 N N Macrococcus caseolyticus 1194NR_074941 clade_547 N N Streptomyces sp. 1 AIP_2009 1890 FJ176782clade_548 N N Streptomyces sp. SD 524 1892 EU544234 clade_548 N NStreptomyces sp. SD 528 1893 EU544233 clade_548 N N Streptomycesthermoviolaceus 1895 NR_027616 clade_548 N N Borrelia afzelii 388ABCU01000001 clade_549 N OP Borrelia crocidurae 390 DQ057990 clade_549 NOP Borrelia duttonii 391 NC_011229 clade_549 N OP Borrelia hermsii 393AY597657 clade_549 N OP Borrelia hispanica 394 DQ057988 clade_549 N OPBorrelia persica 395 HM161645 clade_549 N OP Borrelia recurrentis 396AF107367 clade_549 N OP Borrelia spielmanii 398 ABKB01000002 clade_549 NOP Borrelia turicatae 399 NC_008710 clade_549 N OP Borrelia valaisiana400 ABCY01000002 clade_549 N OP Providencia alcalifaciens 1586ABXW01000071 clade_55 N N Providencia rettgeri 1587 AM040492 clade_55 NN Providencia rustigianii 1588 AM040489 clade_55 N N Providenciastuartii 1589 AF008581 clade_55 N N Treponema pallidum 1932 CP001752clade_550 N OP Treponema phagedenis 1934 AEFH01000172 clade_550 N NTreponema sp. clone DDKL_4 1939 Y08894 clade_550 N N Acholeplasmalaidlawii 17 NR_074448 clade_551 N N Mycoplasma putrefaciens 1323 U26055clade_551 N N Mycoplasmataceae genomo sp. 1325 DQ003614 clade_551 N N P1oral clone MB1_G23 Spiroplasma insolitum 1750 NR_025705 clade_551 N NCollinsella intestinalis 660 ABXH02000037 clade_553 N N Collinsellastercoris 661 ABXJ01000150 clade_553 N N Collinsella tanakaei 662AB490807 clade_553 N N Caminicella sporogenes 458 NR_025485 clade_554 NN Acidaminococcus fermentans 21 CP001859 clade_556 N N Acidaminococcusintestini 22 CP003058 clade_556 N N Acidaminococcus sp. D21 23ACGB01000071 clade_556 N N Phascolarctobacterium 1462 NR_026111clade_556 N N faecium Phascolarctobacterium sp. YIT 1463 AB490812clade_556 N N 12068 Phascolarctobacterium 1464 AB490811 clade_556 N Nsuccinatutens Acidithiobacillus ferrivorans 25 NR_074660 clade_557 N NXanthomonadaceae bacterium 2015 EU313791 clade_557 N N NML 03_0222Catabacter hongkongensis 494 AB671763 clade_558 N N Christensenellaminuta 512 AB490809 clade_558 N N Clostridiales bacterium oral 536AY207065 clade_558 N N clone P4PA_66 P1 Clostridiales bacterium oral 537GQ422712 clade_558 N N taxon 093 Heliobacterium modesticaldum 1000NR_074517 clade_560 N N Alistipes indistinctus 130 AB490804 clade_561 NN Bacteroidales bacterium ph8 257 JN837494 clade_561 N N CandidatusSulcia muelleri 475 CP002163 clade_561 N N Cytophaga xylanolytica 742FR733683 clade_561 N N Flavobacteriaceae genomo sp. 884 AY278614clade_561 N N C1 Gramella forsetii 958 NR_074707 clade_561 N NSphingobacterium faecium 1740 NR_025537 clade_562 N N Sphingobacteriummizutaii 1741 JF708889 clade_562 N N Sphingobacterium multivorum 1742NR_040953 clade_562 N N Sphingobacterium spiritivorum 1743 ACHA02000013clade_562 N N Jonquetella anthropi 1017 ACOO02000004 clade_563 N NPyramidobacter piscolens 1614 AY207056 clade_563 N N Synergistes genomosp. C1 1904 AY278615 clade_563 N N Synergistes sp. RMA 14551 1905DQ412722 clade_563 N N Synergistetes bacterium 1906 GQ258968 clade_563 NN ADV897 Candidatus Arthromitus sp. 474 NR_074460 clade_564 N NSFB_mouse_Yit Gracilibacter thermotolerans 957 NR_043559 clade_564 N NBrachyspira aalborgi 404 FM178386 clade_565 N N Brachyspira sp. HIS3 406FM178387 clade_565 N N Brachyspira sp. HIS4 407 FM178388 clade_565 N NBrachyspira sp. HIS5 408 FM178389 clade_565 N N Adlercreutziaequolifaciens 97 AB306661 clade_566 N N Coriobacteriaceae bacterium 678CAEM01000062 clade_566 N N JC110 Coriobacteriaceae bacterium 679JN837493 clade_566 N N phl Cryptobacterium curtum 740 GQ422741 clade_566N N Eggerthella sinensis 779 AY321958 clade_566 N N Eggerthella sp.1_3_56FAA 780 ACWN01000099 clade_566 N N Eggerthella sp. HGA1 781AEXR01000021 clade_566 N N Eggerthella sp. YY7918 782 AP012211 clade_566N N Gordonibacter pamelaeae 680 AM886059 clade_566 N N Gordonibacterpamelaeae 956 FP929047 clade_566 N N Slackia equolifaciens 1732 EU377663clade_566 N N Slackia exigua 1733 ACUX01000029 clade_566 N N Slackiafaecicanis 1734 NR_042220 clade_566 N N Slackia heliotrinireducens 1735NR_074439 clade_566 N N Slackia isoflavoniconvertens 1736 AB566418clade_566 N N Slackia piriformis 1737 AB490806 clade_566 N N Slackia sp.NATTS 1738 AB505075 clade_566 N N Chlamydiales bacterium NS13 506JN606075 clade_567 N N Victivallaceae bacterium NML 2003 FJ394915clade_567 N N 080035 Victivallis vadensis 2004 ABDE02000010 clade_567 NN Streptomyces griseus 1889 NR_074787 clade_573 N N Streptomyces sp. SD511 1891 EU544231 clade_573 N N Streptomyces sp. SD 534 1894 EU544232clade_573 N N Cloacibacillus evryensis 530 GQ258966 clade_575 N NDeferribacteres sp. oral clone 743 AY349370 clade_575 N N JV001Deferribacteres sp. oral clone 745 AY349372 clade_575 N N JV023Synergistetes bacterium 1907 GQ258969 clade_575 N N LBVCM1157Synergistetes bacterium oral 1909 GU410752 clade_575 N N taxon 362Synergistetes bacterium oral 1910 GU430992 clade_575 N N taxon D48Peptococcus sp. oral clone 1439 AY349389 clade_576 N N JM048Helicobacter winghamensis 999 ACDO01000013 clade_577 N N Wolinellasuccinogenes 2014 BX571657 clade_577 N N Olsenella genomo sp. C1 1368AY278623 clade_578 N N Olsenella profusa 1369 FN178466 clade_578 N NOlsenella sp. F0004 1370 EU592964 clade_578 N N Olsenella sp. oral taxon809 1371 ACVE01000002 clade_578 N N Olsenella uli 1372 CP002106clade_578 N N Nocardiopsis dassonvillei 1356 CP002041 clade_579 N NPeptococcus niger 1438 NR_029221 clade_580 N N Peptococcus sp. oraltaxon 1440 GQ422727 clade_580 N N 167 Akkermansia muciniphila 118CP001071 clade_583 N N Opitutus terrae 1373 NR_074978 clade_583 N NClostridiales bacterium oral 538 HM099644 clade_584 N N taxon F32Leptospira borgpetersenii 1161 NC_008508 clade_585 N OP Leptospirabroomii 1162 NR_043200 clade_585 N OP Leptospira interrogans 1163NC_005823 clade_585 N OP Methanobrevibacter 1213 NR_044789 clade_587 N Ngottschalkii Methanobrevibacter millerae 1214 NR_042785 clade_587 N NMethanobrevibacter oralis 1216 HE654003 clade_587 N N Methanobrevibacterthaueri 1219 NR_044787 clade_587 N N Methanobrevibacter smithii 1218ABYV02000002 clade_588 N N Deinococcus radiodurans 746 AE000513clade_589 N N Deinococcus sp. R_43890 747 FR682752 clade_589 N N Thermusaquaticus 1923 NR_025900 clade_589 N N Actinomyces sp. c109 81 AB167239clade_590 N N Syntrophomonadaceae 1912 AY341821 clade_590 N N genomo sp.P1 Anaerobaculum 141 ACJX02000009 clade_591 N N hydrogeniformansMicrocystis aeruginosa 1246 NC_010296 clade_592 N N Prochlorococcusmarinus 1567 CP000551 clade_592 N N Methanobrevibacter 1208 NR_028779clade_593 N N acididurans Methanobrevibacter 1209 NR_042783 clade_593 NN arboriphilus Methanobrevibacter curvatus 1210 NR_044796 clade_593 N NMethanobrevibacter cuticularis 1211 NR_044776 clade_593 N NMethanobrevibacter filiformis 1212 NR_044801 clade_593 N NMethanobrevibacter woesei 1220 NR_044788 clade_593 N N Roseiflexuscastenholzii 1642 CP000804 clade_594 N N Methanobrevibacter olleyae 1215NR_043024 clade_595 N N Methanobrevibacter 1217 NR_042784 clade_595 N Nruminantium Methanobrevibacter wolinii 1221 NR_044790 clade_595 N NMethanosphaera stadtmanae 1222 AY196684 clade_595 N N Chloroflexi genomosp. P1 511 AY331414 clade_596 N N Halorubrum lipolyticum 992 AB477978clade_597 N N Methanobacterium formicicum 1207 NR_025028 clade_597 N NAcidilobus saccharovorans 24 AY350586 clade_598 N N Hyperthermusbutylicus 1006 CP000493 clade_598 N N Ignicoccus islandicus 1011 X99562clade_598 N N Metallosphaera sedula 1206 D26491 clade_598 N NThermofilum pendens 1922 X14835 clade_598 N N Prevotella melaninogenica1506 CP002122 clade_6 N N Prevotella sp. ICM1 1520 HQ616385 clade_6 N NPrevotella sp. oral clone 1535 AY349393 clade_6 N N FU048 Prevotella sp.oral clone GI030 1537 AY349395 clade_6 N N Prevotella sp. SEQ116 1526JN867246 clade_6 N N Streptococcus anginosus 1787 AECT01000011 clade_60N N Streptococcus milleri 1812 X81023 clade_60 N N Streptococcus sp.16362 1829 JN590019 clade_60 N N Streptococcus sp. 69130 1832 X78825clade_60 N N Streptococcus sp. AC15 1833 HQ616356 clade_60 N NStreptococcus sp. CM7 1839 HQ616373 clade_60 N N Streptococcus sp. OBRC61847 HQ616352 clade_60 N N Burkholderia ambifaria 442 AAUZ01000009clade_61 N OP Burkholderia cenocepacia 443 AAHI01000060 clade_61 N OPBurkholderia cepacia 444 NR_041719 clade_61 N OP Burkholderia mallei 445CP000547 clade_61 N Category-B Burkholderia multivorans 446 NC_010086clade_61 N OP Burkholderia oklahomensis 447 DQ108388 clade_61 N OPBurkholderia pseudomallei 448 CP001408 clade_61 N Category-BBurkholderia rhizoxinica 449 HQ005410 clade_61 N OP Burkholderia sp. 383450 CP000151 clade_61 N OP Burkholderia xenovorans 451 U86373 clade_61 NOP Prevotella buccae 1488 ACRB01000001 clade_62 N N Prevotella genomosp. P8 oral 1498 DQ003622 clade_62 N N clone MB3_P13 Prevotella sp. oralclone 1536 AY349394 clade_62 N N FW035 Prevotella bivia 1486ADFO01000096 clade_63 N N Prevotella disiens 1494 AEDO01000026 clade_64N N Bacteroides faecis 276 GQ496624 clade_65 N N Bacteroides fragilis279 AP006841 clade_65 N N Bacteroides nordii 285 NR_043017 clade_65 N NBacteroides salyersiae 292 EU136690 clade_65 N N Bacteroides sp. 1_1_14293 ACRP01000155 clade_65 N N Bacteroides sp. 1_1_6 295 ACIC01000215clade_65 N N Bacteroides sp. 2_1_56FAA 298 ACWI01000065 clade_65 N NBacteroides sp. AR29 316 AF139525 clade_65 N N Bacteroides sp. B2 317EU722733 clade_65 N N Bacteroides thetaiotaomicron 328 NR_074277clade_65 N N Actinobacillus minor 45 ACFT01000025 clade_69 N NHaemophilus parasuis 978 GU226366 clade_69 N N Vibrio cholerae 1996AAUR01000095 clade_71 N Category-B Vibrio fluvialis 1997 X76335 clade_71N Category-B Vibrio furnissii 1998 CP002377 clade_71 N Category-B Vibriomimicus 1999 ADAF01000001 clade_71 N Category-B Vibrio parahaemolyticus2000 AAWQ01000116 clade_71 N Category-B Vibrio sp. RC341 2001ACZT01000024 clade_71 N Category-B Vibrio vulnificus 2002 AE016796clade_71 N Category-B Lactobacillus acidophilus 1067 CP000033 clade_72 NN Lactobacillus amylolyticus 1069 ADNY01000006 clade_72 N NLactobacillus amylovorus 1070 CP002338 clade_72 N N Lactobacilluscrispatus 1078 ACOG01000151 clade_72 N N Lactobacillus delbrueckii 1080CP002341 clade_72 N N Lactobacillus helveticus 1088 ACLM01000202clade_72 N N Lactobacillus kalixensis 1094 NR_029083 clade_72 N NLactobacillus kefiranofaciens 1095 NR_042440 clade_72 N N Lactobacillusleichmannii 1098 JX986966 clade_72 N N Lactobacillus sp. 66c 1120FR681900 clade_72 N N Lactobacillus sp. KLDS 1.0701 1122 EU600905clade_72 N N Lactobacillus sp. KLDS 1.0712 1130 EU600916 clade_72 N NLactobacillus sp. oral clone 1136 AY349383 clade_72 N N HT070Lactobacillus ultunensis 1139 ACGU01000081 clade_72 N N Prevotellaintermedia 1502 AF414829 clade_81 N N Prevotella nigrescens 1511AFPX01000069 clade_81 N N Prevotella pallens 1515 AFPY01000135 clade_81N N Prevotella sp. oral taxon 310 1551 GQ422737 clade_81 N N Prevotellagenomo sp. C1 1495 AY278624 clade_82 N N Prevotella sp. CM38 1519HQ610181 clade_82 N N Prevotella sp. oral taxon 317 1552 ACQH01000158clade_82 N N Prevotella sp. SG12 1527 GU561343 clade_82 N N Prevotelladenticola 1493 CP002589 clade_83 N N Prevotella genomo sp. P7 oral 1497DQ003620 clade_83 N N clone MB2_P31 Prevotella histicola 1501 JN867315clade_83 N N Prevotella multiformis 1508 AEWX01000054 clade_83 N NPrevotella sp. JCM 6330 1522 AB547699 clade_83 N N Prevotella sp. oralclone GI059 1539 AY349397 clade_83 N N Prevotella sp. oral taxon 7821555 GQ422745 clade_83 N N Prevotella sp. oral taxon G71 1559 GU432180clade_83 N N Prevotella sp. SEQ065 1524 JN867234 clade_83 N N Prevotellaveroralis 1565 ACVA01000027 clade_83 N N Bacteroides acidifaciens 266NR_028607 clade_85 N N Bacteroides cellulosilyticus 269 ACCH01000108clade_85 N N Bacteroides clarus 270 AFBM01000011 clade_85 N NBacteroides eggerthii 275 ACWG01000065 clade_85 N N Bacteroidesoleiciplenus 286 AB547644 clade_85 N N Bacteroides pyogenes 290NR_041280 clade_85 N N Bacteroides sp. 315_5 300 FJ848547 clade_85 N NBacteroides sp. 31SF15 301 AJ583248 clade_85 N N Bacteroides sp. 31SF18302 AJ583249 clade_85 N N Bacteroides sp. 35AE31 303 AJ583244 clade_85 NN Bacteroides sp. 35AE37 304 AJ583245 clade_85 N N Bacteroides sp.35BE34 305 AJ583246 clade_85 N N Bacteroides sp. 35BE35 306 AJ583247clade_85 N N Bacteroides sp. WH2 324 AY895180 clade_85 N N Bacteroidessp. XB12B 325 AM230648 clade_85 N N Bacteroides stercoris 327ABFZ02000022 clade_85 N N Actinobacillus 46 NR_074857 clade_88 N Npleuropneumoniae Actinobacillus ureae 48 AEVG01000167 clade_88 N NHaemophilus aegyptius 969 AFBC01000053 clade_88 N N Haemophilus ducreyi970 AE017143 clade_88 N OP Haemophilus haemolyticus 973 JN175335clade_88 N N Haemophilus influenzae 974 AADP01000001 clade_88 N OPHaemophilus 975 GU561425 clade_88 N N parahaemolyticus Haemophilusparainfluenzae 976 AEWU01000024 clade_88 N N Haemophilus 977 M75076clade_88 N N paraphrophaemolyticus Haemophilus somnus 979 NC_008309clade_88 N N Haemophilus sp. 70334 980 HQ680854 clade_88 N N Haemophilussp. HK445 981 FJ685624 clade_88 N N Haemophilus sp. oral clone 982AY923117 clade_88 N N ASCA07 Haemophilus sp. oral clone 983 AY923147clade_88 N N ASCG06 Haemophilus sp. oral clone 984 AY005034 clade_88 N NBJ021 Haemophilus sp. oral clone 985 AY005033 clade_88 N N BJ095Haemophilus sp. oral taxon 987 AGRK01000004 clade_88 N N 851 Haemophilussputorum 988 AFNK01000005 clade_88 N N Histophilus somni 1003 AF549387clade_88 N N Mannheimia haemolytica 1195 ACZX01000102 clade_88 N NPasteurella bettyae 1433 L06088 clade_88 N N Moellerella wisconsensis1253 JN175344 clade_89 N N Morganella morganii 1265 AJ301681 clade_89 NN Morganella sp. JB_T16 1266 AJ781005 clade_89 N N Proteus mirabilis1582 ACLE01000013 clade_89 N N Proteus penneri 1583 ABVP01000020clade_89 N N Proteus sp. HS7514 1584 DQ512963 clade_89 N N Proteusvulgaris 1585 AJ233425 clade_89 N N Oribacterium sinus 1374 ACKX01000142clade_90 N N Oribacterium sp. ACB1 1375 HM120210 clade_90 N NOribacterium sp. ACB7 1376 HM120211 clade_90 N N Oribacterium sp. CM121377 HQ616374 clade_90 N N Oribacterium sp. ICM51 1378 HQ616397 clade_90N N Oribacterium sp. OBRC12 1379 HQ616355 clade_90 N N Oribacterium sp.oral taxon 1382 AFIH01000001 clade_90 N N 108 Actinobacillus 44 AY362885clade_92 N N actinomycetemcomitans Actinobacillus succinogenes 47CP000746 clade_92 N N Aggregatibacter 112 CP001733 clade_92 N Nactinomycetemcomitans Aggregatibacter aphrophilus 113 CP001607 clade_92N N Aggregatibacter segnis 114 AEPS01000017 clade_92 N N Averyelladalhousiensis 194 DQ481464 clade_92 N N Bisgaard Taxon 368 AY683487clade_92 N N Bisgaard Taxon 369 AY683489 clade_92 N N Bisgaard Taxon 370AY683491 clade_92 N N Bisgaard Taxon 371 AY683492 clade_92 N N Buchneraaphidicola 440 NR_074609 clade_92 N N Cedecea davisae 499 AF493976clade_92 N N Citrobacter amalonaticus 517 FR870441 clade_92 N NCitrobacter braakii 518 NR_028687 clade_92 N N Citrobacter farmeri 519AF025371 clade_92 N N Citrobacter freundii 520 NR_028894 clade_92 N NCitrobacter gillenii 521 AF025367 clade_92 N N Citrobacter koseri 522NC_009792 clade_92 N N Citrobacter murliniae 523 AF025369 clade_92 N NCitrobacter rodentium 524 NR_074903 clade_92 N N Citrobacter sedlakii525 AF025364 clade_92 N N Citrobacter sp. 30_2 526 ACDJ01000053 clade_92N N Citrobacter sp. KMSI_3 527 GQ468398 clade_92 N N Citrobacterwerkmanii 528 AF025373 clade_92 N N Citrobacter youngae 529 ABWL02000011clade_92 N N Cronobacter malonaticus 737 GU122174 clade_92 N NCronobacter sakazakii 738 NC_009778 clade_92 N N Cronobacter turicensis739 FN543093 clade_92 N N Enterobacter aerogenes 786 AJ251468 clade_92 NN Enterobacter asburiae 787 NR_024640 clade_92 N N Enterobactercancerogenus 788 Z96078 clade_92 N N Enterobacter cloacae 789 FP929040clade_92 N N Enterobacter cowanii 790 NR_025566 clade_92 N NEnterobacter hormaechei 791 AFHR01000079 clade_92 N N Enterobacter sp.247BMC 792 HQ122932 clade_92 N N Enterobacter sp. 638 793 NR_074777clade_92 N N Enterobacter sp. JC163 794 JN657217 clade_92 N NEnterobacter sp. SCSS 795 HM007811 clade_92 N N Enterobacter sp. TSE38796 HM156134 clade_92 N N Enterobacteriaceae bacterium 797 ADCU01000033clade_92 N N 9_2_54FAA Enterobacteriaceae bacterium 798 AJ489826clade_92 N N CF01Ent_1 Enterobacteriaceae bacterium 799 AY538694clade_92 N N Smarlab 3302238 Escherichia albertii 824 ABKX01000012clade_92 N N Escherichia coli 825 NC_008563 clade_92 N Category-BEscherichia fergusonii 826 CU928158 clade_92 N N Escherichia hermannii827 HQ407266 clade_92 N N Escherichia sp. 1_1_43 828 ACID01000033clade_92 N N Escherichia sp. 4_1_40B 829 ACDM02000056 clade_92 N NEscherichia sp. B4 830 EU722735 clade_92 N N Escherichia vulneris 831NR_041927 clade_92 N N Ewingella americana 877 JN175329 clade_92 N NHaemophilus genomo sp. P2 971 DQ003621 clade_92 N N oral clone MB3_C24Haemophilus genomo sp. P3 972 DQ003635 clade_92 N N oral clone MB3_C38Haemophilus sp. oral clone 986 AY349380 clade_92 N N JM053 Hafnia alvei989 DQ412565 clade_92 N N Klebsiella oxytoca 1024 AY292871 clade_92 N OPKlebsiella pneumoniae 1025 CP000647 clade_92 N OP Klebsiella sp. AS101026 HQ616362 clade_92 N N Klebsiella sp. Co9935 1027 DQ068764 clade_92N N Klebsiella sp. enrichment 1036 HM195210 clade_92 N N culture cloneSRC_DSD25 Klebsiella sp. OBRC7 1028 HQ616353 clade_92 N N Klebsiella sp.SP_BA 1029 FJ999767 clade_92 N N Klebsiella sp. SRC_DSD1 1033 GU797254clade_92 N N Klebsiella sp. SRC_DSD11 1030 GU797263 clade_92 N NKlebsiella sp. SRC_DSD12 1031 GU797264 clade_92 N N Klebsiella sp.SRC_DSD15 1032 GU797267 clade_92 N N Klebsiella sp. SRC_DSD2 1034GU797253 clade_92 N N Klebsiella sp. SRC_DSD6 1035 GU797258 clade_92 N NKlebsiella variicola 1037 CP001891 clade_92 N N Kluyvera ascorbata 1038NR_028677 clade_92 N N Kluyvera cryocrescens 1039 NR_028803 clade_92 N NLeminorella grimontii 1159 AJ233421 clade_92 N N Leminorella richardii1160 HF558368 clade_92 N N Pantoea agglomerans 1409 AY335552 clade_92 NN Pantoea ananatis 1410 CP001875 clade_92 N N Pantoea brenneri 1411EU216735 clade_92 N N Pantoea citrea 1412 EF688008 clade_92 N N Pantoeaconspicua 1413 EU216737 clade_92 N N Pantoea septica 1414 EU216734clade_92 N N Pasteurella dagmatis 1434 ACZR01000003 clade_92 N NPasteurella multocida 1435 NC_002663 clade_92 N N Plesiomonasshigelloides 1469 X60418 clade_92 N N Raoultella ornithinolytica 1617AB364958 clade_92 N N Raoultella planticola 1618 AF129443 clade_92 N NRaoultella terrigena 1619 NR_037085 clade_92 N N Salmonella bongori 1683NR_041699 clade_92 N Category-B Salmonella enterica 1672 NC_011149clade_92 N Category-B Salmonella enterica 1673 NC_011205 clade_92 NCategory-B Salmonella enterica 1674 DQ344532 clade_92 N Category-BSalmonella enterica 1675 ABEH02000004 clade_92 N Category-B Salmonellaenterica 1676 ABAK02000001 clade_92 N Category-B Salmonella enterica1677 NC_011080 clade_92 N Category-B Salmonella enterica 1678 EU118094clade_92 N Category-B Salmonella enterica 1679 NC_011094 clade_92 NCategory-B Salmonella enterica 1680 AE014613 clade_92 N Category-BSalmonella enterica 1682 ABFH02000001 clade_92 N Category-B Salmonellaenterica 1684 ABEM01000001 clade_92 N Category-B Salmonella enterica1685 ABAM02000001 clade_92 N Category-B Salmonella typhimurium 1681DQ344533 clade_92 N Category-B Salmonella typhimurium 1686 AF170176clade_92 N Category-B Serratia fonticola 1718 NR_025339 clade_92 N NSerratia liquefaciens 1719 NR_042062 clade_92 N N Serratia marcescens1720 GU826157 clade_92 N N Serratia odorifera 1721 ADBY01000001 clade_92N N Serratia proteamaculans 1722 AAUN01000015 clade_92 N N Shigellaboydii 1724 AAKA01000007 clade_92 N Category-B Shigella dysenteriae 1725NC_007606 clade_92 N Category-B Shigella flexneri 1726 AE005674 clade_92N Category-B Shigella sonnei 1727 NC_007384 clade_92 N Category-BTatumella ptyseos 1916 NR_025342 clade_92 N N Trabulsiella guamensis1925 AY373830 clade_92 N N Yersinia aldovae 2019 AJ871363 clade_92 N OPYersinia aleksiciae 2020 AJ627597 clade_92 N OP Yersinia bercovieri 2021AF366377 clade_92 N OP Yersinia enterocolitica 2022 FR729477 clade_92 NCategory-B Yersinia frederiksenii 2023 AF366379 clade_92 N OP Yersiniaintermedia 2024 AF366380 clade_92 N OP Yersinia kristensenii 2025ACCA01000078 clade_92 N OP Yersinia mollaretii 2026 NR_027546 clade_92 NOP Yersinia pestis 2027 AE013632 clade_92 N Category-A Yersiniapseudotuberculosis 2028 NC_009708 clade_92 N OP Yersinia rohdei 2029ACCD01000071 clade_92 N OP Yokenella regensburgei 2030 AB273739 clade_92N N Conchiformibius kuhniae 669 NR_041821 clade_94 N N Morococcuscerebrosus 1267 JN175352 clade_94 N N Neisseria bacilliformis 1328AFAY01000058 clade_94 N N Neisseria cinerea 1329 ACDY01000037 clade_94 NN Neisseria flavescens 1331 ACQV01000025 clade_94 N N Neisseriagonorrhoeae 1333 CP002440 clade_94 N OP Neisseria lactamica 1334ACEQ01000095 clade_94 N N Neisseria macacae 1335 AFQE01000146 clade_94 NN Neisseria meningitidis 1336 NC_003112 clade_94 N OP Neisseria mucosa1337 ACDX01000110 clade_94 N N Neisseria pharyngis 1338 AJ239281clade_94 N N Neisseria polysaccharea 1339 ADBE01000137 clade_94 N NNeisseria sicca 1340 ACKO02000016 clade_94 N N Neisseria sp. KEM232 1341GQ203291 clade_94 N N Neisseria sp. oral clone AP132 1344 AY005027clade_94 N N Neisseria sp. oral strain B33KA 1346 AY005028 clade_94 N NNeisseria sp. oral taxon 014 1347 ADEA01000039 clade_94 N N Neisseriasp. TM10_1 1343 DQ279352 clade_94 N N Neisseria subflava 1348ACEO01000067 clade_94 N N Okadaella gastrococcus 1365 HQ699465 clade_98N N Streptococcus agalactiae 1785 AAJO01000130 clade_98 N NStreptococcus alactolyticus 1786 NR_041781 clade_98 N N Streptococcusaustralis 1788 AEQR01000024 clade_98 N N Streptococcus bovis 1789AEEL01000030 clade_98 N N Streptococcus canis 1790 AJ413203 clade_98 N NStreptococcus constellatus 1791 AY277942 clade_98 N N Streptococcuscristatus 1792 AEVC01000028 clade_98 N N Streptococcus dysgalactiae 1794AP010935 clade_98 N N Streptococcus equi 1795 CP001129 clade_98 N NStreptococcus equinus 1796 AEVB01000043 clade_98 N N Streptococcusgallolyticus 1797 FR824043 clade_98 N N Streptococcus genomo sp. C1 1798AY278629 clade_98 N N Streptococcus genomo sp. C2 1799 AY278630 clade_98N N Streptococcus genomo sp. C3 1800 AY278631 clade_98 N N Streptococcusgenomo sp. C4 1801 AY278632 clade_98 N N Streptococcus genomo sp. C51802 AY278633 clade_98 N N Streptococcus genomo sp. C6 1803 AY278634clade_98 N N Streptococcus genomo sp. C7 1804 AY278635 clade_98 N NStreptococcus genomo sp. C8 1805 AY278609 clade_98 N N Streptococcusgordonii 1806 NC_009785 clade_98 N N Streptococcus infantarius 1807ABJK02000017 clade_98 N N Streptococcus infantis 1808 AFNN01000024clade_98 N N Streptococcus intermedius 1809 NR_028736 clade_98 N NStreptococcus lutetiensis 1810 NR_037096 clade_98 N N Streptococcusmassiliensis 1811 AY769997 clade_98 N N Streptococcus mitis 1813AM157420 clade_98 N N Streptococcus oligofermentans 1815 AY099095clade_98 N N Streptococcus oralis 1816 ADMV01000001 clade_98 N NStreptococcus parasanguinis 1817 AEKM01000012 clade_98 N N Streptococcuspasteurianus 1818 AP012054 clade_98 N N Streptococcus peroris 1819AEVF01000016 clade_98 N N Streptococcus pneumoniae 1820 AE008537clade_98 N N Streptococcus porcinus 1821 EF121439 clade_98 N NStreptococcus 1822 FJ827123 clade_98 N N pseudopneumoniae Streptococcuspseudoporcinus 1823 AENS01000003 clade_98 N N Streptococcus pyogenes1824 AE006496 clade_98 N OP Streptococcus ratti 1825 X58304 clade_98 N NStreptococcus sanguinis 1827 NR_074974 clade_98 N N Streptococcussinensis 1828 AF432857 clade_98 N N Streptococcus sp. 2_1_36FAA 1831ACOI01000028 clade_98 N N Streptococcus sp. 2285_97 1830 AJ131965clade_98 N N Streptococcus sp. ACS2 1834 HQ616360 clade_98 N NStreptococcus sp. AS20 1835 HQ616366 clade_98 N N Streptococcus sp.BS35a 1836 HQ616369 clade_98 N N Streptococcus sp. C150 1837ACRI01000045 clade_98 N N Streptococcus sp. CM6 1838 HQ616372 clade_98 NN Streptococcus sp. ICM10 1840 HQ616389 clade_98 N N Streptococcus sp.ICM12 1841 HQ616390 clade_98 N N Streptococcus sp. ICM2 1842 HQ616386clade_98 N N Streptococcus sp. ICM4 1844 HQ616387 clade_98 N NStreptococcus sp. ICM45 1843 HQ616394 clade_98 N N Streptococcus sp.M143 1845 ACRK01000025 clade_98 N N Streptococcus sp. M334 1846ACRL01000052 clade_98 N N Streptococcus sp. oral clone 1849 AY923121clade_98 N N ASB02 Streptococcus sp. oral clone 1850 DQ272504 clade_98 NN ASCA03 Streptococcus sp. oral clone 1851 AY923116 clade_98 N N ASCA04Streptococcus sp. oral clone 1852 AY923119 clade_98 N N ASCA09Streptococcus sp. oral clone 1853 AY923123 clade_98 N N ASCB04Streptococcus sp. oral clone 1854 AY923124 clade_98 N N ASCB06Streptococcus sp. oral clone 1855 AY923127 clade_98 N N ASCC04Streptococcus sp. oral clone 1856 AY923128 clade_98 N N ASCC05Streptococcus sp. oral clone 1857 DQ272507 clade_98 N N ASCC12Streptococcus sp. oral clone 1858 AY923129 clade_98 N N ASCD01Streptococcus sp. oral clone 1859 AY923130 clade_98 N N ASCD09Streptococcus sp. oral clone 1860 DQ272509 clade_98 N N ASCD10Streptococcus sp. oral clone 1861 AY923134 clade_98 N N ASCE03Streptococcus sp. oral clone 1862 AY953253 clade_98 N N ASCE04Streptococcus sp. oral clone 1863 DQ272510 clade_98 N N ASCE05Streptococcus sp. oral clone 1864 AY923135 clade_98 N N ASCE06Streptococcus sp. oral clone 1865 AY923136 clade_98 N N ASCE09Streptococcus sp. oral clone 1866 AY923137 clade_98 N N ASCE10Streptococcus sp. oral clone 1867 AY923138 clade_98 N N ASCE12Streptococcus sp. oral clone 1868 AY923140 clade_98 N N ASCF05Streptococcus sp. oral clone 1869 AY953255 clade_98 N N ASCF07Streptococcus sp. oral clone 1870 AY923142 clade_98 N N ASCF09Streptococcus sp. oral clone 1871 AY923145 clade_98 N N ASCG04Streptococcus sp. oral clone 1872 AY005042 clade_98 N N BW009Streptococcus sp. oral clone 1873 AY005044 clade_98 N N CH016Streptococcus sp. oral clone 1874 AY349413 clade_98 N N GK051Streptococcus sp. oral clone 1875 AY349414 clade_98 N N GM006Streptococcus sp. oral clone 1876 AY207051 clade_98 N N P2PA_41 P2Streptococcus sp. oral clone 1877 AY207064 clade_98 N N P4PA_30 P4Streptococcus sp. oral taxon 1878 AEEP01000019 clade_98 N N 071Streptococcus sp. oral taxon 1879 GU432132 clade_98 N N G59Streptococcus sp. oral taxon 1880 GU432146 clade_98 N N G62Streptococcus sp. oral taxon 1881 GU432150 clade_98 N N G63Streptococcus suis 1882 FM252032 clade_98 N N Streptococcus thermophilus1883 CP000419 clade_98 N N Streptococcus salivarius 1826 AGBV01000001clade_98 N N Streptococcus uberis 1884 HQ391900 clade_98 N NStreptococcus urinalis 1885 DQ303194 clade_98 N N Streptococcusvestibularis 1886 AEKO01000008 clade_98 N N Streptococcus viridans 1887AF076036 clade_98 N N Synergistetes bacterium oral 1908 GU227192clade_98 N N clone 03 5 D05

TABLE X4 Spore-forming Bacterial Species Alkaliphilus metalliredigensAmmonifex degensii Anaerofustis stercorihominis Anaerostipes caccaeAnaerotruncus colihominis Bacillus amyloliquefaciens Bacillus anthracisBacillus cellulosilyticus Bacillus cereus Bacillus clausii Bacilluscoagulans Bacillus cytotoxicus Bacillus halodurans Bacilluslicheniformis Bacillus pumilus Bacillus subtilis Bacillus thuringiensisBacillus weihenstephanensis Blautia hansenii Brevibacillus brevisBryantella formatexigens Caldicellulosiruptor saccharolyticus CandidatusDesulforudis audaxviato Carboxydibrachium pacificum Carboxydothermushydrogenoformans Clostridium acetobutylicum Clostridium asparagiformeClostridium bartlettii Clostridium beijerinckii Clostridium bolteaeClostridium botulinum A str. ATCC 19397 Clostridium botulinum B str.Eklund 17B Clostridium butyricum pathogenic E4 str. BoNT BL5262Clostridium Carboxidivorans Clostridium cellulolyticum Clostridiumcellulovorans Clostridium difficile Clostridium hathewayi Clostridiumhylemonae Clostridium kluyveri Clostridium leptum Clostridiummethylpentosum Clostridium nexile Clostridium novyi NT Clostridiumpapyrosolvens Clostridium perfringens Clostridium phytofermentans ISDgClostridium scindens Clostridium sp. 7_2_43FAA Clostridium sporogenesClostridium tetani Clostridium thermocellum Coprococcus comesDesulfotomaculum reducens Dorea longicatena Eubacterium eligensEubacterium hallii Eubacterium rectale Eubacterium ventriosumFaecalibacterium prausnitzii Geobacillus kaustophilus Geobacillus sp.G11MC16 Geobacillus thermodenitrificans Heliobacterium modesticaldumLysinibacillus sphaericus Oceanobacillus iheyensis Paenibacillus sp.JDR-2 Pelotomaculum thermopropionicum Roseburia intestinalisRuminococcus bromii Ruminococcus gnavus Ruminococcus obeum Ruminococcustorques Subdoligranulum variabile Symbiobacterium thermophilumThermoanaerobacter italicus Thermoanaerobacter tengcongensisThermoanaerobacterium thermosaccharolyticum Thermosinus carboxydivorans

TABLE 2 Species isolated from ethanol treated spore preparationpreparation before (left) and after (right) CsCl gradient step ethanoltreated ethanol treated, spore gradient purified Isolates preparationspore preparation Bacillus coagulans 7 2 Blautia luti 1 1 Blautia sp 1413 Blautia wexlerae 3 1 Ruminococcus obeum 4 2 Clostridiales sp 1 2Clostridium aerotolerans 1 2 Clostridium disporicum 0 1 Clostridium sp 11 Clostridium symbiosum 0 1 Dorea longicatena 8 6 Eubacteriumcellulosolvens 1 0 Eubacterium ventriosum 2 2 Gemmiger formicilis 0 1Robinsoniella peoriensis 0 1 Roseburia hominis 3 6 Roseburiaintestinalis 9 7 Ruminococcus sp 5 2 Syntrophococcus sucromutans 1 1Turicibacter sanguinis 3 4 Clostridiales sp 7 9 Clostridium bartlettii 811 Clostridium irregulare 0 1 Clostridium sordellii 4 6 Lachnospiraceaesp 1 0

TABLE 3 Mortality and weight change in mice challenged with C. difficilewith or without ethanol treated, spore product treatment. mortality %weight change Test article (n = 10) on Day 3 vehicle (negative control)20% −10.5% Donor feces (positive control) 0  −0.1% EtOH-treated feces 1×0    2.3% EtOH-treated feces 0.1× 0    2.4% EtOH-treated feces 0.01× 0  −3% heat-treated feces 0    0.1%

TABLE 4 16S rDNA identified spore formind species from picked colonyplates. Treatment Species No. isolates  70 deg 1 h Clostridium_celatum 4 70 deg 1 h Clostridium_clostridioform 1  70 deg 1 hClostridium_hylemonae 1  70 deg 1 h Clostridium_paraputrificum 3  70 deg1 h Clostridium_sp_D5 1  70 deg 1 h Clostridium_symbiosum 1  80 deg 1 hClostridium_bartlettii 6  80 deg 1 h Clostridium_butyricum 1  80 deg 1 hClostridium_paraputrificum 5  80 deg 1 h Coprobacillus_sp_D7 1  80 deg 1h Eubacterium_sp_WAL_14571 1  80 deg 1 h Ruminococcus_bromii 1  90 deg 1h Clostridium_butyricum 1  90 deg 10 min Ruminococcus_bromii 1  90 deg10 min Anaerotruncus_colihominis 2  90 deg 10 min Clostridium_bartlettii1 100 deg 10 min Ruminococcus_bromii 1

TABLE 5 Spore-forming species identified in ethanol treated or heattreated samples and not identified in untreated samples isolatedisolated isolated from from from EtOH- heat- Species untreated treatedtreated Acetivibrio ethanolgignens X Anaerofustis stercorihominis XBacillus anthracis X Bacillus horti X Bacillus licheniformis X Bacillusnealsonii X Bacillus pumilus X Bacillus sp. BT1B_CT2 X Bacillusthuringiensis X Bacteroides galacturonicus X (phylogenetically inClostridiales) Bacteroides pectinophilus X (phylogenetically inClostridiales) Blautia wexlerae X X Brachyspira pilosicoli XBrevibacillus parabrevis X Clostridium aldenense X Clostridiumbeijerinckii X Clostridium carnis X Clostridium celatum X Clostridiumfavososporum X Clostridium hylemonae X Clostridium irregulare XClostridium methylpentosum X Clostridium sp. D5 X X Clostridium sp.L2-50 X Clostridium sp. MT4 E X Clostridium sp. NML 04A032 X Clostridiumsp. SS2/1 X Clostridium sp. YIT 12069 X Clostridium stercorarium XClostridium xylanolyticum X Coprococcus sp. ART55/1 X Deferribacteressp. oral clone JV006 X Desulfitobacterium frappieri X Eubacteriumcallanderi X Eubacterium siraeum X Exiguobacterium acetylicum X Gemmigerformicilis X Lachnospira multipara X Lachnospira pectinoschiza XRoseburia faecalis X Ruminococcus albus X

TABLE 6 Donor A, 45 species in 374 EtOH-resistant colonies sequenced OTUAnaerostipes_sp_3_2_56FAA Bacillus_anthracis Bacillus_cereusBacillus_thuringiensis Blautia_producta Blautia_sp_M25Clostridiales_sp_SSC_2 Clostridium_aldenense Clostridium_bartlettiiClostridium_bolteae Clostridium_celatum Clostridium_disporicumClostridium_ghonii Clostridium_hathewayi Clostridium_lactatifermentansClostridium_mayombei Clostridium_orbiscindens Clostridium_paraputrificumClostridium_perfringens Clostridium_sordellii Clostridium_stercorariumClostridium_straminisolvens Clostridium_tertium Coprobacillus_sp_D7Coprococcus_catus Deferribacteres_sp_oral_clone_JV006Dorea_formicigenerans Eubacterium_rectale Eubacterium_siraeumEubacterium_sp_WAL_14571 Eubacterium_ventriosum Flexistipes_sinusarabiciFulvimonas_sp_NML_060897 Lachnospiraceae_bacterium_2_1_58FAALachnospiraceae_bacterium_3_1_57FAA Lachnospiraceae_bacterium_A4Lachnospiraceae_bacterium_oral_taxon_F15 Moorella_thermoaceticaRoseburia_faecalis Roseburia_hominis Ruminococcus_albusRuminococcus_bromii Ruminococcus_gnavus Ruminococcus_sp_5_1_39BFAARuminococcus_torques

TABLE 7 Donor B, 26 species in 195 EtOH-resistant colonies sequenced OTUBacillus_horti Blautia_wexlerae Chlamydiales_bacterium_NS11Clostridiales_sp_SSC_2 Clostridium_bartlettii Clostridium_celatumClostridium_disporicum Clostridium_ghonii Clostridium_oroticumClostridium_paraputrificum Clostridium_perfringens Clostridium_sordelliiClostridium_sp_L2_50 Clostridium_sp_MT4_E Clostridium_straminisolvensCoprococcus_sp_ART55_1 Eubacterium_callanderi Eubacterium_rectaleEubacterium_ruminantium Gemmiger_formicilis Lachnospira_pectinoschizaRuminococcus_albus Ruminococcus_gnavus Ruminococcus_obeumRuminococcus_sp_5_1_39BFAA Ruminococcus_sp_K_1

TABLE 8 Donor C, 39 species in 416 EtOH-resistant colonies sequenced OTUBacteroides _(—) galacturonicus Bacteroides _(—) pectinophilus Blautia_(—) producta Blautia_sp_M25 Blautia _(—) wexleraeClostridiales_sp_SS3_4 Clostridiales_sp_SSC_2 Clostridium _(—)bartlettii Clostridium _(—) citroniae Clostridium _(—) disporicumClostridium _(—) indolis Clostridium _(—) orbiscindens Clostridium _(—)paraputrificum Clostridium _(—) sordellii Clostridium_sp_NML_04A032Clostridium_sp_SS2_1 Clostridium _(—) straminisolvens Clostridium _(—)viride Clostridium _(—) xylanolyticum Coprobacillus_sp_D7 Dorea _(—)longicatena Eubacterium _(—) rectale Eubacterium _(—) ventriosumHydrogenoanaerobacterium _(—) saccharovorans Lachnospira _(—) multiparaLachnospira _(—) pectinoschiza Lachnospiraceae_bacterium_A4Oscillibacter_sp_G2 Pseudoflavonifractor _(—) capillosus Roseburia _(—)hominis Roseburia _(—) intestinalis Ruminococcus _(—) albus Ruminococcus_(—) lactaris Ruminococcus _(—) obeum Ruminococcus_sp_5_1_39BFAARuminococcus_sp_K_1 Ruminococcus_torques Syntrophococcus _(—)sucromutans

TABLE 9 Donor D, 12 species in 118 EtOH-resistant colonies sequenced OTUBlautia _(—) luti Blautia _(—) wexlerae Brachyspira _(—) pilosicoliClostridium _(—) paraputrificum Collinsella _(—) aerofaciensCoprobacillus_sp_D7 Desulfitobacterium _(—) frappieri Eubacterium _(—)rectale Moorella _(—) thermoacetica Ruminococcus _(—) gnavusRuminococcus _(—) obeum Ruminococcus_sp_K_1

TABLE 10 Donor E, 11 species in 118 EtOH-resistant colonies sequencedOTU Blautia _(—) luti Blautia _(—) wexlerae Brachyspira _(—) pilosicoliClostridium _(—) paraputrificum Coprobacillus_sp_D7 Desulfitobacterium_(—) frappieri Eubacterium _(—) rectale Moorella _(—) thermoaceticaRuminococcus _(—) gnavus Ruminococcus _(—) obeum Ruminococcus_sp_K_1

TABLE 11 Donor F, 54 OTUs in 768 EtOH-resistant colonies sequenced OTUAnaerofustis _(—) stercorihominis Anaerostipes_sp_3_2_56FAA Bacillus_(—) nealsonii Bacillus_sp_BT1B_CT2 Blautia _(—) producta Butyrivibrio_(—) crossotus Clostridiales_bacterium_SY8519 Clostridiales_sp_1_7_47Clostridium _(—) aldenense Clostridium _(—) bartlettii Clostridium _(—)bolteae Clostridium _(—) butyricum Clostridium _(—) citroniaeClostridium _(—) clostridioforme Clostridium _(—) disporicum Clostridium_(—) favososporum Clostridium _(—) glycolicum Clostridium _(—) hathewayiClostridium _(—) indolis Clostridium _(—) leptum Clostridium _(—)mayombei Clostridium _(—) nexile Clostridium _(—) orbiscindensClostridium _(—) sordellii Clostridium_sp_7_2_43FAA Clostridium_sp_D5Clostridium_sp_M62_1 Clostridium_sp_NML_04A032 Clostridium _(—)spiroforme Clostridium _(—) symbiosum Clostridium _(—) tertiumCoprobacillus_sp_29_1 Coprobacillus_sp_D7 Eubacterium _(—) contortumEubacterium _(—) desmolans Eubacterium _(—) ramulus Exiguobacterium _(—)acetylicum Faecalibacterium _(—) prausnitziiLachnospiraceae_bacterium_2_1_58FAA Lachnospiraceae_bacterium_3_1_57FAALachnospiraceae_bacterium_5_1_57FAA Lachnospiraceae_bacterium_6_1_63FAALachnospiraceae_bacterium_oral_taxon_F15 Marvinbryantia _(—)formatexigens Mycoplasma _(—) amphoriforme Oscillibacter_sp_G2Pseudoflavonifractor _(—) capillosus Ruminococcus _(—) gnavusRuminococcus _(—) hansenii Ruminococcus _(—) obeumRuminococcus_sp_5_1_39BFAA Ruminococcus_sp_ID8 Turicibacter _(—)sanguinis

TABLE 12 Organisms grown from ethanol treated spore population onvarious media (See Example 5 for full media names and references). totalnumber unique % unique Media reads OTUs OTUs M2GSC 93 33 0.35 M-BHI 6626 0.39 Sweet B 74 23 0.31 GAM fructose 44 18 0.41 M2 mannitol 39 170.44 M2 soluble starch 62 16 0.26 M2 lactate 43 14 0.33 GAM FOS/Inulin52 14 0.27 EYA 29 13 0.45 Mucin 19 12 0.63 M2 lactose 32 12 0.38 BHISaz1/ge2 35 12 0.34 BHIS CInM az1/ge2 24 11 0.46 GAM mannitol 41 11 0.27BBA 29 10 0.34 Sulfite-polymyxin milk 48 9 0.19 Noack-Blaut Eubacteriumagar 12 4 0.33 742 total analyzed

TABLE 13 Species identified as germinable and sporulatable by colonypicking Sweet GAM + B + FOS/ FOS/ Sweet OTU BBA inulin M2GSC Inulin GAMTotal Blautia producta 1 1 Clostridium 4 1 5 bartlettii Clostridiumbolteae 2 5 1 8 Clostridium 5 5 botulinum Clostridium 37 43 8 1 33 122butyricum Clostridium celatum 4 1 5 Clostridium 1 1 2 clostridioformeClostridium 26 26 22 33 50 157 disporicum Clostridium 4 9 14 27glycolicum Clostridium 2 2 4 mayombei Clostridium 8 8 33 16 6 71paraputrificum Clostridium sordellii 14 14 Clostridium sp. 1 1 7_2_43FAAClostridium 3 3 symbiosum Clostridium tertium 1 1 2 (blank) 2 31 33Totals 92 92 92 92 92 460

TABLE 15 Results of the prophylaxis mouse model and dosing informationfor the germinable, and sporulatable fractions. Clinical score is basedon a combined phenotypic assessment of the mouse's health on a scale of0-4 in several areas including appearance (0-2 pts based on normal,hunched, piloerection, or lethargic), and clinical signs (0-2 pointsbased on normal, wet tail, cold-to-the-touch, or isolation from otheranimals). Average Average Weight on Day Clinical # Deaths 3 RelativeScore Test Article Dose by Day 6 to Day −1 on Day 3 Vehicle NA 10 0.72NA Naive NA 0 1.03 0 Donor B 0.2 mL of 10% 1 0.91 0.11 fecal suspensionsuspension Donor A 8.99*10{circumflex over ( )}7 Spore 0 1.02 0 SporePrep Equivalents/dose germinable Donor A 7.46*10{circumflex over ( )}7Spore 0 0.99 0 Spore Prep Equivalents/dose Sporulatable

TABLE 16 Bacterial OTUs associated with engraftment and ecologicalaugmentation and establishment of a more diverse microbial ecology inpatients treated with an ethanol treated spore preparation. DominantPhylo- Spore OTU in genetic Forming Augmented OTU Clade OTU EcologyBacteroides sp. 2_1_22 clade38 N Y Streptococcus anginosus clade60 NPrevotella intermedia clade81 N Prevotella nigrescens clade81 NOribacterium sp. ACB7 clade90 N Prevotella salivae clade104 NBacteroides intestinalis clade171 N Y Bifidobacterium dentium clade172 NAlcaligenes faecalis clade183 N Rothia dentocariosa clade194 NPeptoniphilus lacrimalis clade291 N Anaerococcus sp. gpac155 clade294 NSutterella stercoricanis clade302 N Y Bacteroides sp. 3_1_19 clade335 NY Parabacteroides goldsteinii clade335 N Bacteroides dorei clade378 N YBacteroides massiliensis clade378 N Lactobacillus iners clade398 NGranulicatella adiacens clade460 N Eggerthella sp. 1_3_56FAA clade477 NGordonibacter pamelaeae clade477 N Finegoldia magna clade509 NActinomyces nasicola clade523 N Streptobacillus moniliformis clade532 NOscillospira guilliermondii clade540 N Orientia tsutsugamushi clade541 NChristensenella minuta clade558 N Clostridium oroticum clade96 YClostridium sp. D5 clade96 Y Clostridium glycyrrhizinilyticum clade147 YCoprococcus comes clade147 Y Ruminococcus lactaris clade147 YRuminococcus torques clade147 Y Y Clostridiales sp. SS3/4 clade246 YClostridium hylemonae clade260 Y Clostridium aerotolerans clade269 YClostridium asparagiforme clade300 Y Y Clostridium sp. M62/1 clade300 YClostridium symbiosum clade300 Y Lachnospiraceae genomosp. C1 clade300 YBlautia sp. M25 clade304 Y Y Blautia stercoris clade304 Y Ruminococcushansenii clade304 Y Ruminococcus obeum clade304 Y Ruminococcus sp.5_1_39BFAA clade304 Y Bryantella formatexigens clade309 Y Eubacteriumcellulosolvens clade309 Y Clostridium sp. HGF2 clade351 Y Clostridiumbartlettii clade354 Y Clostridium bifermentans clade354 Y Clostridiumglycolicum clade354 Y Eubacterium tenue clade354 Y Dorea formicigeneransclade360 Y Dorea longicatena clade360 Y Lachnospiraceae bacteriumclade360 Y 2_1_46FAA Lachnospiraceae bacterium clade360 Y Y 9_1_43BFAARuminococcus gnavus clade360 Y Clostridium hathewayi clade362 Y Blautiahydrogenotrophica clade368 Y Clostridiaceae bacterium END-2 clade368 YRoseburia faecis clade369 Y Roseburia hominis clade370 Y Roseburiaintestinalis clade370 Y Eubacterium sp. WAL 14571 clade384 YErysipelotrichaceae bacterium clade385 Y 5_2_54FAA Eubacterium biformeclade385 Y Eubacterium dolichum clade385 Y Coprococcus catus clade393 YAcetivibrio ethanolgignens clade396 Y Anaerosporobacter mobilis clade396Y Bacteroides pectinophilus clade396 Y Eubacterium hallii clade396 YEubacterium xylanophilum clade396 Y Anaerostipes caccae clade408 YClostridiales bacterium clade408 Y 1_7_47FAA Clostridium aldenenseclade408 Y Clostridium citroniae clade408 Y Eubacterium hadrum clade408Y Y Acetanaerobacterium elongatum clade439 Y Faecalibacteriumprausnitzii clade478 Y Gemmiger formicilis clade478 Y Y Eubacteriumramulus clade482 Y Lachnospiraceae bacterium clade483 Y 3_1_57FAA_CT1Lachnospiraceae bacterium A4 clade483 Y Y Lachnospiraceae bacterium DJFclade483 Y VP30 Holdemania filiformis clade485 Y Clostridiumorbiscindens clade494 Y Pseudoflavonifractor capillosus clade494 YRuminococcaceae bacterium D16 clade494 Y Acetivibrio cellulolyticusclade495 Y Eubacterium limosum clade512 Y Anaerotruncus colihominisclade516 Y Clostridium methylpentosum clade516 Y Clostridium sp. YIT12070 clade516 Y Hydrogenoanaerobacterium clade516 Y saccharovoransEubacterium ventriosum clade519 Y Eubacterium eligens clade522 YLachnospira pectinoschiza clade522 Y Lactobacillus rogosae clade522 Y YClostridium leptum clade537 Y Eubacterium coprostanoligenes clade537 YRuminococcus bromii clade537 Y Clostridium viride clade540 YButyrivibrio crossotus clade543 Y Coprococcus eutactus clade543 YEubacterium ruminantium clade543 Y Eubacterium rectale clade568 Y YRoseburia inulinivorans clade568 Y Butyricicoccus pullicaecorum clade572Y Eubacterium desmolans clade572 Y Papillibacter cinnamivorans clade572Y Sporobacter termitidis clade572 Y Clostridium lactatifermentansclade576 Y

TABLE 18 Reduction in the opportunistic pathogen or pathobiont load byethanol treated spores. Pre- treatment Day 5 Day 14 Day 25 Klebsiella (%of total reads) 20.27% 1.32% 7.62% 0.00% Fusobacterium (% total ofreads) 19.14% 3.01% 0.01% 0.00%

TABLE 19 Changes in Enterobacteria as a function of treatment measuredon Simmons Citrate Agar Pretreatment titer Day 25 titer Patient Organism(cfu/g) (cfu/g) 1 Klebsiella pneumoniae 9 × 10⁶ 1 × 10³ 1 Klebsiella sp.Co9935 4 × 10⁶ 1 × 10³ 1 Escherichia coli 7 × 10⁶ 1 × 10⁶ 2 Klebsiellasp. Co9935 4 × 10⁶ 1 × 10³ 4 Klebsiella pneumoniae 3 × 10⁸ <1 × 10⁴  4Klebsiella sp. Co9935 6 × 10⁷ <1 × 10⁴  5 Klebsiella pneumoniae 1 × 10⁶<1 × 10⁴ 

TABLE 20 Augmentation of Bacteroides as a function of bacterialcomposition treatment of Patient 1 Pretreatment titer Day 25 titer MediaBacteroides species (cfu/g) (cfu/g) BBE B. fragilis group <2 × 10⁴ 3 ×10⁸  PFA All Bacteroides <2 × 10⁷ 2 × 10¹⁰

TABLE 21 Bacteroides spp. post-treatment with the ethanol treated sporepreparation based full-length 16S rDNA sequences of isolated strains %of total Bacteroides cfu Species (1.58E10 cfu/g) Bacteroides sp. 4_1_36 63% Bacteroides cellulosilyticus  14% Bacteroides sp. 1_1_30  14%Bacteroides uniformis  4.8% Bacteroides ovatus  1.7% Bacteroides dorei0.91% Bacteroides xylanisolvens 0.83% Bacteroides sp. 3_1_19 0.23%

TABLE 22 Titers (in cfu/g) of imipenem-resistant M. morganii, P.rettgeri and P. pennerii from Patients B, D & E Patient OrganismPretreatment titer Day 28 titer * Patient 2 M. morganii 1 × 10⁴  6 × 10²Patient 2 P. rettgeri 9 × 10³ <5 × 10¹ Patient 4 M. morganii 2 × 10⁴ <5× 10¹ Patient 4 P. pennerii 2 × 10⁴ <5 × 10¹ Patient 5 M. morganii 5 ×10³ <5 × 10¹ * Limit of detection based on plating 200 uL of 10% wt/volsuspension is 5 × 10¹

TABLE YYY Species identified as germinable by 16S colony pick approachClostridium _(—) paraputrificum Clostridium _(—) disporicum Clostridium_(—) glycolicum Clostridium _(—) bartlettii Clostridium _(—) butyricumRuminococcus _(—) bromii Lachnospiraceae_bacterium_2_1_58FAA Eubacterium_(—) hadrum Turicibacter _(—) sanguinisLachnospiraceae_bacterium_oral_taxon_F15 Clostridium _(—) perfringensClostridium _(—) bifermentans Roseburia_sp_11SE37 Clostridium _(—)quinii Ruminococcus _(—) lactaris Clostridium _(—) botulinum Clostridium_(—) tyrobutyricum Blautia _(—) hansenii Clostridium _(—) kluyveriClostridium_sp_JC122 Clostridium _(—) hylemonae Clostridium _(—) celatumClostridium _(—) straminisolvens Clostridium _(—) orbiscindens Roseburia_(—) cecicola Eubacterium _(—) tenue Clostridium_sp_7_2_43FAALachnospiraceae_bacterium_4_1_37FAA Eubacterium _(—) rectale Clostridium_(—) viride Ruminococcus_sp_K_1 Clostridium _(—) symbiosum Ruminococcus_(—) torques Clostridium _(—) algidicarnis

TABLE ZZZ Species identified as sporulatable by 16S NGS approachClostridium _(—) paraputrificum Clostridium _(—) bartlettiiLachnospiraceae_bacterium_2_1_58FAA Clostridium _(—) disporicumRuminococcus _(—) bromii Eubacterium _(—) hadrum Clostridium _(—)butyricum Roseburia_sp_11SE37 Clostridium _(—) perfringens Clostridium_(—) glycolicum Clostridium _(—) hylemonae Clostridium _(—) orbiscindensRuminococcus _(—) lactaris Clostridium _(—) symbiosumLachnospiraceae_bacterium_oral_taxon_F15 Blautia _(—) hanseniiTuricibacter _(—) sanguinis Clostridium _(—) straminisolvens Clostridium_(—) botulinum Lachnospiraceae_bacterium_4_1_37FAA Roseburia _(—)cecicola Ruminococcus_sp_K_1 Clostridium _(—) bifermentans Eubacterium_(—) rectale Clostridium _(—) quinii Clostridium _(—) viride Clostridium_(—) kluyveri Clostridium _(—) tyrobutyricum Oscillibacter_sp_G2Clostridium_sp_JC122 Lachnospiraceae_bacterium_3_1_57FAA Clostridium_(—) aldenense Ruminococcus _(—) torques Clostridium_sp_7_2_43FAAClostridium _(—) celatum Eubacterium_sp_WAL_14571 Eubacterium _(—) tenueLachnospiraceae_bacterium_5_1_57FAA Clostridium _(—) clostridioformeClostridium_sp_YIT_12070 Blautia_sp_M25 Anaerostipes _(—) caccaeRoseburia _(—) inulinivorans Clostridium_sp_D5 Clostridium _(—)asparagiforme Coprobacillus_sp_D7 Clostridium_sp_HGF2 Clostridium _(—)citroniae Clostridium _(—) difficile Oscillibacter _(—) valericigenesClostridium _(—) algidicarnis

1.-99. (canceled)
 100. A composition for treating or reducing a severityof at least one symptom of a gastrointestinal disease, disorder orcondition associated with a dysbiosis in a subject, the compositioncomprising a purified population of bacterial spores in an amounteffective to populate a gastrointestinal tract in the subject and acapsule, wherein the bacterial spores belong to clade 396 and comprise a16S sequence that is at least 95% identical to SEQ ID NO:6; and thecomposition is derived from a fecal material subjected to ethanoltreatment or heat treatment; and the composition is substantiallydepleted of a residual habitat product of the fecal material; and thebacterial spores are not detectable in the fecal material before theethanol treatment or the heat treatment.
 101. The composition of claim100, wherein the bacterial spores comprise a 16S sequence that is atleast 97% identical to SEQ ID NO:6.
 102. The composition of claim 100,wherein the bacterial spores comprise a 16S sequence that is 100%identical to SEQ ID NO:6.
 103. The composition of claim 100, wherein thecomposition comprises at least 1×10⁴ colony forming units of thepurified population of bacterial spores per dose of the composition.104. The composition of claim 100, wherein the composition comprises anamount of the purified population of bacterial spores effective toaugment growth in the subject's gastrointestinal tract of at least onebacteria not detectable in the composition or to modulate microbiotadiversity after administration of the composition compared to themicrobiota diversity present in the subject's gastrointestinal tractprior to administration of the composition.
 105. The composition ofclaim 100, wherein the subject is a human.
 106. The composition of claim100, wherein the gastrointestinal disease, disorder or condition isselected from the group consisting of Clostridium difficile-induceddiarrhea, irritable bowel syndrome (IBS), infection or colonization witha pathogen or pathobiont including a drug resistant pathogen orpathobiont, colitis, a metabolic disorder, and Crohn's disease.
 107. Thecomposition of claim 100, wherein the gastrointestinal disease, disorderor condition is Clostridium difficile-induced diarrhea.
 108. Thecomposition of claim 100, wherein the fecal material is a 10 to 20%fecal suspension.
 109. The composition of claim 100, wherein the fecalmaterial is obtained from a validated mammalian donor subject not havinga detectable level of a pathogen or a pathobiont prior to production ofthe fecal material.
 110. The composition of claim 100, wherein the fecalmaterial is obtained from a validated mammalian donor subject not havinga detectable level of a pathogen or a pathobiont prior to production ofthe fecal material.
 111. The composition of claim 100, wherein the fecalmaterial is obtained from a validated mammalian donor subject not havinga detectable level of a a blood-borne pathogen or a fecal bacterialpathogen prior to production of the fecal material.
 112. The compositionof claim 100, wherein the composition is derived from fecal materialsubjected to ethanol treatment.
 113. The composition of claim 100,wherein the composition is derived from fecal material subjected totreatment with 30-90% ethanol.
 114. The composition of claim 100,wherein the composition is derived from fecal material subjected totreatment with 50-70% ethanol.
 115. The composition of claim 100,wherein the composition is derived from fecal material subjected totreatment with 50% ethanol.
 116. The composition of claim 100, whereinthe composition is derived from fecal material subjected to ethanoltreatment and the ethanol treatment comprises a. forming a 10% w/vsuspension of human fecal material; b. mixing the suspension withabsolute ethanol in a 1:1 ratio; c. incubating the suspension of (b); d.collecting the bacterial spores in the suspension; e. adding glycerol tothe bacterial spores of (d); and f. storing the composition at −80degrees Celsius.
 117. The composition of claim 100, wherein thecomposition is derived from fecal material subjected to heat treatmentand the heat treatment comprises a. forming a 10% w/v suspension ofhuman fecal material in PBS; b. incubating the suspension of (a) at 80degrees Celsius for 30 minutes, thereby forming a heat treatedsuspension; c. adding glycerol to the heat treated suspension; and d.storing the composition at −80 degrees Celsius.
 118. The composition ofclaim 100, wherein the residual habitat product is an abiotic material,a human or animal cell, a virus, a fungus, or a mycoplasma.
 119. Thecomposition of claim 100, wherein, using genomic and/or microbiologicalassay methods, the bacterial spores are not detectable in the fecalmaterial before the ethanol treatment or the heat treatment.
 120. Thecomposition of claim 100, wherein, using qPCR, the bacterial spores arenot detectable in the fecal material before the ethanol treatment or theheat treatment.
 121. The composition of claim 100, wherein the bacterialspores are not detectable in the fecal material before the ethanoltreatment or the heat treatment as assayed by full-length 16S sequencingof bacterial colonies grown and isolated from the fecal material beforethe ethanol or heat treatment.
 122. The composition of claim 100,wherein the bacterial spores are not detectable in the fecal materialbefore the ethanol treatment or the heat treatment using an assay with adetectable limit of 20 cfu/ml.
 123. The composition of claim 100,comprising a delayed release capsule.
 124. The composition of claim 100,formulated for oral administration.
 125. The composition of claim 100,further comprising an antibiotic.
 126. The composition of claim 100,further comprising at least one species of germinable bacterial sporesselected from the group consisting of Bacillus licheniformis, Bacilluspumilus, Clostridium hylemonae, Clostridium methypentosum, Clostridiumsp YIT 12069, Anaerofustis stercorihominis, Bacillus anthracis, Bacillushorti, Bacillus nealsonii, Bacillus sp. BT1B_CT2, Bacillusthuringiensis, Bacteroides galacturonicus, Bacteroides pectinophilus,Brachyspira pilosicoli, Clostridium aldenense, Clostridium beijerinckii,Clostridium carnis, Clostridium celatum, Clostridium favososporum,Clostridium irregulare, Clostridium sp. L2-50, Clostridium sp. MT4 E,Clostridium sp. NML 04A032, Clostridium sp. SS2/1, Clostridiumstercorarium, Clostridium xylanolyticum, Coprococcus sp. ART55/1,Deferribacteres sp. oral clone JV006, Desulfitobacterium frappieri,Eubacterium callanderi, Eubacterium siraeum, Exiguobacterium acetylicum,Gemmiger formicilis, Lachnospira multipara, Lachnospira pectinoschiza,Roseburia faecalis, and Ruminococcus albus.